Endothelial nitric oxide synthase (eNOS) decreases following inflammatory stimulation. As a master regulator of endothelial homeostasis, maintaining optimal eNOS levels is important during cardiovascular events. However, little is known regarding the mechanism of eNOS protection. In this study, we demonstrate a regulatory role for endothelial expression of 2′–5′ oligoadenylate synthetase-like 1 (OASL1) in maintaining eNOS mRNA stability during athero-prone conditions and consider its clinical implications. A lack of endothelial Oasl1 accelerated plaque progression, which was preceded by endothelial dysfunction, elevated vascular inflammation, and decreased NO bioavailability following impaired eNOS expression. Mechanistically, knockdown of PI3K/Akt signaling-dependent OASL expression increased Erk1/2 and NF-κB activation and decreased NOS3 (gene name for eNOS) mRNA expression through upregulation of the negative regulatory, miR-584, whereas a miR-584 inhibitor rescued the effects of OASL knockdown. These results suggest that OASL1/OASL regulates endothelial biology by protecting NOS3 mRNA and targeting miR-584 represents a rational therapeutic strategy for eNOS maintenance in vascular disease.

Although arginase II (ArgII) is abundant in mitochondria, Ca 2+ -accumulating organelles, the relationship between ArgII activity and Ca 2+ translocation into mitochondria and the regulation of cytosolic Ca 2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca 2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca 2+ ]m as measured by CoCl 2 -quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII −/− mice. Conversely, [Ca 2+ ]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca 2+ ]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca 2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca 2+ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE −/− +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca 2+ ]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE −/− +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE −/− +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca 2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.

No abstract

Arginase II reciprocally regulates endothelial nitric oxide synthase (eNOS) through a p32-dependent Ca2+ control. We investigated the signaling pathway of arginase II-dependent eNOS phosphorylation. Western blot analysis was applied for examining protein activation and [Ca2+]c was analyzed by microscopic and FACS analyses. Nitric oxide (NO) and reactive oxygen species (ROS) productions were measured using specific fluorescent dyes under microscopy. NO signaling pathway was tested by measuring vascular tension. Following arginase II downregulation by chemical inhibition or gene knockout (KO, ArgII−/−), increased eNOS phosphorylation at Ser1177 and decreased phosphorylation at Thr495 was depend on p38 MAPK activation, which induced by CaMKII activation through p32-dependent increase in [Ca2+]c. The protein amount of p32 negatively regulated p38 MAPK activation. p38 MAPK contributed to Akt-induced eNOS phosphorylation at Ser1177 that resulted in accelerated NO production and reduced reactive oxygen species production in aortic endothelia. In vascular tension assay, p38 MAPK inhibitor decreased acetylcholine-induced vasorelaxation responses and increased phenylephrine-dependent vasoconstrictive responses. In ApoE−/− mice fed a high cholesterol diet, arginase II inhibition restored p32/CaMKII/p38 MAPK/Akt/eNOS signaling cascade that was attenuated by p38 MAPK inhibition. Here, we demonstrated a novel signaling pathway contributing to understanding of the relationship between arginase II, endothelial dysfunction, and atherogenesis.