Because of the high levels of reactive oxygen and nitrogen species (ROS/RNS) present in diseased arteries, the function of endothelium-derived nitric oxide (7NO) is decreased, and this contributes to the growth of vascular lesions. Because of this association, the studies sponsored by this grant have sought to identify key proteins that are physiologically regulated by 7NO, and whose function is impaired by ROS/RNS. In the last period of funding, we identified the sarcoplasmic reticulum Ca2+ ATPase (SERCA) in vascular smooth muscle cells (SMC) to be stimulated by low levels of 7NO-derived RNS that formed glutathione (GSH) adducts most abundantly on SERCA cysteine-674 (C674). We showed that the S-glutathiolation of this reactive thiol increased the Ca2+ uptake activity of SERCA and accompanied endothelium-dependent vasodilatation. In addition, using SERCA C674S mutants, we found that the 7NO-dependent S-glutathiolation of C674 was a requirement for 7NO to lower intracellular Ca2+ and inhibit smooth muscle cell migration, a process key to the growth of vascular lesions. Of importance to diabetic vascular disease, preliminary studies show that C674 can be irreversibly oxidized to a large degree in SMC by hyperlipidemia, high glucose, and in insulin-resistant rodents, preventing the 7NO-induced S-glutathiolation and stimulation of Ca2+ uptake. Thus, the SERCA C674 thiol is a key physiological mediator of 7NO action in SMC whose role is prevented by elevated oxidants associated with disease. New studies indicate that redox regulation of SERCA C674 is also essential for VEGF-induced endothelial cell (EC) migration and angiogenesis. To further understand the redox regulation of SERCA in vivo we have worked to obtain unique tools that will enable us to further understand the redox regulation of SERCA in vivo. These include 1) thiol labeling methods that allow quantitative assessment of the redox state of SERCA C674, 2) a sequence-specific antibody that recognizes irreversibly oxidized SERCA C674, and 3) heterozygote SERCA2 C674S knock-in (SKI+/-) mice that preliminary studies show have abnormal 7NO-stimulated SERCA activity and relaxation, and decreased VEGF- and ischemia-induced angiogenesis. This mouse model will enable us to examine the physiological importance of redox-regulation of SERCA in vivo. The 4 aims are: 1) To understand the physiological role of redox-regulation of Ca2+ uptake by SERCA in SMC. 2) To determine redox regulators of SERCA which inhibit or promote arterial injury-induced neointima in vivo. 3) To understand the physiological role of SERCA in redox regulation of EC and angiogenesis. 4) To determine if oxidation of SERCA C674 contributes to atherogenesis by limiting the normal action of 7NO on SERCA.
Vascular disease, particularly that caused by diabetes mellitus, is initiated and promoted by the impaired ability of nitric oxide (7NO) released by endothelial cells to regulate vascular contraction, migration, and proliferation. The studies funded by this proposal have identified the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) to be stimulated by 7NO via its ability to introduce glutathione SERCA adducts, thereby lowering intracellular Ca2+ concentrations and regulating cell function. Our proposed studies intend to demonstrate the role for this mechanism in vivo using mouse models of diabetic vascular disease, including a mouse that expresses a mutant SERCA that lacks the key thiol.
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