RICERCA ITALIANA     

Research program

ADVANCED TOOLS OF MOLECULAR IMAGING AND GENETIC ENGINEERING TO STUDY DISTRIBUTION AND DYNAMICS OF CELL MAGNESIUM: NOVEL APPROACHES TO LINK MAGNESIUM HOMEOSTASIS AND CELL FUNCTIONS.

University Co-ordinator

Università degli Studi di MILANO - SCIENZE PRECLINICHE "L.I.T.A. DI VIALBA" - ()

Research Unit Leader

Jeanette Anne Marie Maier

Description

Endothelial cells are very sensitive to different concentrations of extracellular Mg (Maier, 2004 a and b, Ferré, 2007). Even though epidemiological, clinical and experimental evidences link low Mg to endothelial dysfunction, crucial in the onset of cardiovascular diseases (Maier, 2003), our knowledge of Mg cellular homeostasis is still scattered and incomplete. Biochemical and molecular data hint at the existence of specific control mechanisms in the membrane that regulate Mg influx and efflux, as well as the possibility that the cation is distributed within different cellular compartments. In human aortic endothelial cells a study has reported an increase of intracellular Mg in response to elevation of extracellular Mg within a short time (2-10 min), but no molecular mechanisms have been unravelled (Zhang, 1997). Recently, the expression of TRPM-7 in endothelial cells has been demonstrated (Yao, 2005).
We propose to determine the intracellular concentrations and distribution of Mg, with a particular attention to ionized Mg, in endothelial cells by a novel approach of molecular imaging. We will correlate these results with endothelial functions using cultured human endothelial cells genetically modified to overexpress or downregulate TRPM-7. The project is articulated in three tasks.
Task I (months 1-8): generation of the experimental model to study. We routinely isolate and culture endothelial cells of different origin. In this project, we plan to utilize endothelial cells derived from the umbilical vein (HUVEC, clone C obtained from ATCC), which are a good model to study endothelial biology. Part of our results on the effects of extracellular Mg have been obtained using these cells (Maier, 2004 a and b, Ferré, 2007). Preliminary experiments have demonstrated that HUVEC express TRPM-7. We propose to transfect the cells with expression vectors containing TRPM-7 or its mutant deleted of the kinase region, kindly provided by dr. Mazur (Clermont Ferrand, France). The deletion mutant will be used to understand the role of the a-kinase in modulating the activity of the channel. After selection, transfectants will be characterized by western blot using anti-TRPM-7 antibodies (Abcam). We also plan to silence TRPM-7 using siRNA. We have successfully utilized this approach (Castiglioni, 2007), also in HUVEC (Leidi, in preparation). Using a lipid-based system for siRNA transfection, we will transiently transfect HUVEC with three different sequences of siRNA (QIAGEN Inc.); as controls, scrambled siRNA will be utilized. After 24, 48 and 72 h, we will use two independent assays to test siRNA activity and specificity. First, we will evaluate the amounts of TRPM-7 by western analysis. Second, we will use fluorescence digital imaging to test Mg transmembrane transport (see below; in collaboration with the U.O in Bologna and Rome). We also plan to utilize vectors for stable transfection of the siRNA in HUVEC. After selection and propagation of the clones, they will be assessed for the amounts of TRPM-7. Previous studies reported that cultured cells rendered TRPM7-deficient via cre-lox–mediated destruction of the TRPM7 gene, undergo growth arrest and die after a few days in culture (Schmitz C, 2003), while transiently silenced smooth muscle cells remained viable for 48-72 h (He, 2005). In case HUVEC do not survive to a permanent silencing of TRPM-7, we will continue our studies with transient siRNA. Alternatively, stable transfected clones which partially inhibit TRPM-7 translation will be used.
These genetically modified HUVEC will allow to understand the role of TRPM-7 in modulating Mg fluxes and its subcellular distribution. They will also be useful to correlate intracellular Mg to endothelial function.
Task II (months 6-24). Once obtained genetically engineered HUVEC, we plan 1) to measure intracellular Mg by fluorescence digital imaging and 2) to study endothelial function.
i) Intracellular Mg will be measured in collaboration with the U.O. in Bologna and Rome, who synthesized new fluorescent molecules based on diaza-18-crown-6 (Farruggia, 2006). These molecules show a high affinity for Mg and are very specific, since interference from calcium is very low. In addition, they are well tolerated by the cells and allow the detection of rapid intracellular Mg transients. HUVEC overexpressing or silencing TRPM-7 will be cultured under normal conditions or in media containing different concentrations of Mg (0.1, 1.0 and 3.0 mM) for various times (10 and 30 min, 4, 24 and 48 h, reasonable times also in case we have to use a transient siRNA approach). By culturing the cells in different concentrations of Mg, we aim to highlight the influence of extracellular on intracellular Mg. By performing the experiments in kinetics, we plan to understand whether intracellular Mg fluctuations are rapidly induced and maintained thereafter. Since TRPM-7 activity is regulated by cAMP and the activation of protein kinase A (Penner, 2007), we will treat genetically modified HUVEC with forskolin, which activates protein kinase A and measure intracellular Mg. Recently, the enhancement of TRPM-7 activity by shear stress has been demonstrated in epithelial cells (Numata, 2007). Fluid shear stress plays a role in regulating vessel caliber and endothelial function. In particular, laminar shear stress promotes endothelial cell quiescence, whereas oscillatory shear stress promotes endothelial turnover and dysfunction. HUVEC will be subjected to either laminar (15 dyn/cm2) or oscillatory (+/-5 dyn/cm2) shear stress for 24 hours (Tressel, 2007). Intracellular Mg will then be evaluated. These studies will underscore the role of TRPM-7 in Mg homeostasis in endothelial cells and are preliminary to the studies described in task III. They will also clarify whether the a-kinase domain contributes to the regulation of Mg transport.
ii) The behaviour of HUVEC overexpressing or silencing TRPM-7 will be evaluated. Also the cells transfected with the kinase deletion mutant will be studied. At regular intervals and at least for a month, we will evaluate the proliferation rate of the cells using a cell counter. We will also study cell distribution through the different phases of the cell cycle by FACS and the expression of cyclins, cyclin dependent kinases and their inhibitors by western blot. In parallel, we will evaluate whether apoptosis occurs in our cells. We will determine internucleosomic cleavage of the DNA by electrophoresis on agarose gel and caspase activation, by commercially available kits. Since endothelial impairment is associated with cardiovascular diseases, in our experimental model we will study the synthesis of nitric oxide, prostacyclin, cytokines and adhesion molecules, parameters which are measured in patients as markers of endothelial function (Félétou, 2006). We have shown that nitric oxide (NO) synthesis is reduced in HUVEC cultured in low Mg. In our experimental model, we will study the expression of endothelial and inducible NO synthase (NOS) by Northern blot and immunoprecipitation, and the enzymatic activity measuring the conversion of arginine in citrulline. We will also evaluate the synthesis of prostacyclins by ELISA and the expression of cyclo-oxygenase-2 by western analysis.
It is widely accepted that the adhesion of monocytes is an early event in atherogenesis. We will therefore evaluate whether different levels of expression of TRPM-7 modulate endothelial adhesiveness by specific assays in vitro using Cr-labelled monocytes. In the case of a positive result, we will study which adhesion molecules (VCAM, ICAM, E- and P-seletin) are involved by western blot and FACS analysis using specific antibodies. Since 1) low Mg induces the synthesis of cytokines in vivo and in vitro and 2) endothelial cells produce several cytokines and chemokines, we will analyze the cytokine profile in HUVEC overexpressing and silencing TRPM-7 by protein array. Protein array, one emerging class of proteomic technologies, is a potent tool to study the protein profile of specific members of known signal pathways and protein network. We have successfully used this technique to study the cytokine profile of endothelial cells grown under different conditions (Maier 2006, Cotrupi 2005). Briefly, a protein array contains an array of immobilized protein spots. The protein arrays we plan to utilize display antibodies against cytokines, chemokines and angiogenic factors. The membrane will be incubated with either cell extracts or culture media from HUVEC expressing different levels of TRPM-7. The differences in protein profile will be visualized by chemiluminescence. The results obtained will be confirmed by western blot and/or ELISA. Recently, we found that HUVEC cultured in low Mg upregulate PPARgamma, important player in lipid metabolism and atherogenesis. It will be interesting to repeat these experiments in HUVEC overexpressing or silencing TRPM-7. We will first evaluate the amounts of PPARgamma by western blot. To study its transcriptional activity, we will transfect HUVEC with a plasmid containing the luciferase reporter gene under the control of an artificial promoter obtained by fusing four consensus sequences for PPARgamma (the construct is ready and utilized in the lab). We will analyze whether the transcriptional activity of PPARgamma is modulated in HUVEC engineered for TRPM-7 by measuring luciferase activity.
These studies will highlight the role of TRPM-7 in modulating endothelial cell behaviour in vitro. They will also allow to correlate endothelial function to the levels of expression of TRPM-7 and, on the basis of the imaging studies, with the intracellular concentration and distribution of Mg.
Task III (months 12-24). In collaboration with the U.O at C.N.R., HUVEC engineered for TRPM-7 will be analyzed by X-ray microscopy. On the basis of the data obtained by the U.O. in Rome on epithelial cells, we will set up the appropriate conditions to grow the cells (conventional coverslips can not be used because they absorb the radiation), to fix and permeabilize them. Since rigorous analytical techniques for sensitive measurements of intracellular Mg are lacking, this novel and sophisticated approach should yield new insights about Mg concentration and distribution within endothelial cells, thus underscoring the role of TRPM-7 in the homeostasis of Mg in these cells.


The project is feasible since the techniques and the expertises are available in the laboratory. The collaboration with the other U.O. will be fundamental to fulfill all the objectives of the project.