Enzymatic characterization of recombinant rat DDHD2, a soluble diacylglycerol lipase

学位記番号：医博甲1589

Activation of the Sphingosine 1 Phosphate–Rho Pathway in Pterygium and in Ultraviolet-Irradiated Normal Conjunctiva

Sphingosine 1 phosphate (S1P) is a bioactive lipid that regulates cellular activity, including proliferation, cytoskeletal organization, migration, and fibrosis. In this study, the potential relevance of S1P–Rho signaling in pterygium formation and the effects of ultraviolet (UV) irradiation on activation of the S1P/S1P receptor axis and fibrotic responses were investigated in vitro. Expressions of the S1P2, S1P4, and S1P5 receptors were significantly higher in pterygium tissue than in normal conjunctiva, and the concentration of S1P was significantly elevated in the lysate of normal conjunctival fibroblast cell (NCFC) irradiated with UV (UV-NCFCs). RhoA activity was significantly upregulated in pterygium fibroblast cells (PFCs) and UV-NCFCs, and myosin phosphatase–Rho interacting protein (MRIP) was upregulated, and myosin phosphatase target subunit 1 (MYPT1) was downregulated in PFCs. Fibrogenic changes were significantly upregulated in both PFCs and UV-NCFCs compared to NCFCs. We found that the activation of the S1P receptor–Rho cascade was observed in pterygium tissue. Additionally, in vitro examination showed S1P–rho activation and fibrogenic changes in PFCs and UV-NCFCs. S1P elevation and the resulting upregulation of the downstream Rho signaling pathway may be important in pterygium formation; this pathway offers a potential therapeutic target for suppressing pterygium generation

Impaired endothelial barrier function in apolipoprotein M-deficient mice is dependent on sphingosine-1-phosphate receptor 1

Apolipoprotein M (ApoM) transports sphingosine-1-phosphate (S1P) in plasma, and ApoM-deficient mice (Apom(−/−)) have ∼50% reduced plasma S1P levels. There are 5 known S1P receptors, and S1P induces adherens junction formation between endothelial cells through the S1P(1) receptor, which in turn suppresses vascular leak. Increased vascular permeability is a hallmark of inflammation. The purpose of this study was to explore the relationships between vascular leakage in ApoM deficiency and S1P(1) function in normal physiology and in inflammation. Vascular permeability in the lungs was assessed by accumulation of dextran molecules (70 kDa) and was increased ∼40% in Apom(−/−) mice compared to WT (C57Bl6/j) mice. Reconstitution of plasma ApoM/S1P or treatment with an S1P(1) receptor agonist (SEW2871) rapidly reversed the vascular leakage to a level similar to that in WT mice, suggesting that it is caused by decreased plasma levels of S1P and reduced S1P(1) stimulation. In a carrageenan-induced model of inflammation, Apom(−/−) mice had increased vascular leakage compared with that in WT mice. Adenoviral overexpression of ApoM in Apom(−/−) mice decreased the vascular leakage compared to adenoviral overexpression of green fluorescent protein. The study suggests that vascular leakage of albumin-sized particles in ApoM deficiency is S1P- and S1P(1)-dependent and this dependency exacerbates the response to inflammatory stimuli.—Christensen, P. M., Liu, C. H., Swendeman, S. L., Obinata, H., Qvortrup, K., Nielsen, L B., Hla, T., Di Lorenzo, A., Christoffersen, C. Impaired endothelial barrier function in apolipoprotein M-deficient mice is dependent on sphingosine-1-phosphate receptor 1

⁶⁴Cu-ATSM Predicts Efficacy of Carbon Ion Radiotherapy Associated with Cellular Antioxidant Capacity

Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. We addressed this issue by focusing on cellular antioxidant capacity and investigated 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. We found that the carbon ion RBE correlated with 64Cu-ATSM uptake both in vitro and in vivo. High RBE/64Cu-ATSM cells showed greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts; this upregulation of antioxidant systems was associated with downregulation of TCA cycle intermediates. Furthermore, inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) sensitized high RBE/64Cu-ATSM cells to X-rays, thereby reducing RBE values to levels comparable to those in low RBE/64Cu-ATSM cells. These data suggest that the cellular activity of Nrf2-driven antioxidant systems is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. These new findings highlight the potential clinical utility of 64Cu-ATSM imaging to identify high RBE tumors that will benefit from carbon ion radiotherapy

HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation.

The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S1P1 in adult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, was enhanced in mice with endothelial cell-specific deletion of S1pr1 and suppressed in mice with endothelial cell-specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. The chaperones ApoM(+)HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; therefore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM(+)HDL-S1P, but not to albumin-S1P, promoted the formation of a cell surface S1P1-β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-1 abundance. Although S1P bound to either chaperone induced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelial cell-specific deletion of S1pr1 in the hypercholesterolemic Apoe(-/-) mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM(+)HDL to act as a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL

Reduced Fatty Acid Use from CD36 Deficiency Deteriorates Streptozotocin-Induced Diabetic Cardiomyopathy in Mice

Cardiac dysfunction is induced by multifactorial mechanisms in diabetes. Deranged fatty acid (FA) utilization, known as lipotoxicity, has long been postulated as one of the upstream events in the development of diabetic cardiomyopathy. CD36, a transmembrane glycoprotein, plays a major role in FA uptake in the heart. CD36 knockout (CD36KO) hearts exhibit reduced rates of FA transport with marked enhancement of glucose use. In this study, we explore whether reduced FA use by CD36 ablation suppresses the development of streptozotocin (STZ)-induced diabetic cardiomyopathy. We found that cardiac contractile dysfunction had deteriorated 16 weeks after STZ treatment in CD36KO mice. Although accelerated glucose uptake was not reduced in CD36KO-STZ hearts, the total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. The isotopomer analysis with 13C6-glucose revealed that accelerated glycolysis, estimated by enrichment of 13C2-citrate and 13C2-malate, was markedly suppressed in CD36KO-STZ hearts. Levels of ceramides, which are cardiotoxic lipids, were not elevated in CD36KO-STZ hearts compared to wild-type-STZ ones. Furthermore, increased energy demand by transverse aortic constriction resulted in synergistic exacerbation of contractile dysfunction in CD36KO-STZ mice. These findings suggest that CD36KO-STZ hearts are energetically compromised by reduced FA use and suppressed glycolysis; therefore, the limitation of FA utilization is detrimental to cardiac energetics in this model of diabetic cardiomyopathy