Reactive Oxygen Species-induced Cytosolic Calcium(II) Signaling in Endothelial Cells and Involvement of TRPM2

Reactive Oxygen Species (ROS) play a key role in normal physiological processes such as cell proliferation and growth, as well as in pathological processes. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. Oxidative stress is now thought to underlie many cardiovascular diseases. Accumulating evidence also demonstrate that many ROS-induced functional abnormalities in the cardiovascular system are preceded by an elevation of intracellular Ca2+.;In the first part, I compared the Ca2+ responses to ROS between mouse endothelial cells derived from large-sized artey aortas (aortic ECs), and small-sized mesenteric arteries (MAECs). Application of hydrogen peroxide (H2O2) caused an increase in cytosolic Ca 2+ levels ([Ca2+]i) in both cell types. The [Ca2+]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP3) receptors. In addition, treatment of endothelial cells with H2O2 reduced the Ca2+ responses to subsequent challenge of ATP. The decreased Ca2+ responses to ATP were resulted from a pre-depletion of intracellular Ca2+ stores by H2O2. Interestingly, we also found that Ca2+ store depletion was more sensitive to H2O2 treatment in endothelial cells derived from mesenteric arteries than those of derived from aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca2+]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H2O2 but not by hydroxyl radicals. H2O2 made a greater contribution to HX-XO-induced [Ca 2+]i rises in endothelial cells from mesenteric arteries than those from aortas. In summary, H2O2 could induce store Ca2+ release via phospholipase C-IP3 pathway in endothelial cells. Emptying of intracellular Ca2+ stores contributed to the reduced Ca2+ responses to subsequent ATP challenge. Furthermore, the Ca2+ responses in endothelial cells of small-sized arteries were more sensitive to H2O2 than those of large-sized arteries.;In the second part, I used murine heart microvessel endothelial cell line H5V as a model of endothelial cells from small-sized arteries to investigate the role of Melastatin-like transient receptor potential channel 2 (TRPM2) channels in H2O2-induced Ca2+ responses and apoptosis. TRPM2 is an oxidant-sensitive cationic non-selective channel that is expressed in mammalian vascular endothelium. A TRPM2 blocking antibody channel (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the Ca 2+ responses to H2O2. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect in H2O2-induced Ca2+ responses. H 2O2-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect on H2O2-induced apoptotic cell death. In contrast, overexpression of TRPM2 in H5V cells increased the Ca2+ responses to H2O2 and aggravated the apoptotic cell death in response to H2O2. These findings strongly suggest that the TRPM2 channel mediates Ca2+ overload in response to H2O2 and contributes to oxidant-induced apoptotic cell death in vascular endothelial cells. I also examined the downstream cascades of TRPM2 activation and explored whether caspase-3, -8 and -9 were involved in this process. My data indicates that H2O2-induced cell apoptosis through both intrinsic and extrinsic apoptotic pathways, leading to activation of caspases-3. Furthermore, TRPM2 played an essential role in the process. Together, my data suggest that TRPM2 mediates H2 O2-induces endothelial cell death and that down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.