作者: Upchurch, Clint M. ; Merk, Dennis ; Perez-Reyes, Edward ; Raghavan, Shyam S. ; Bochkis, Irina M. ; Yeudall, Scott ; Pavelec, Caitlin M. ; Greulich, Jan ; Scott, Michael M. ; Manjegowda, Mohan ; Leitinger, Norbert

Oxidized phosphatidylcholines (OxPCs) are implicated in chronic tissue damage. Hyperlipidemic LDL-R-–deficient mice transgenic for an OxPC-recognizing IgM fragment (scFv-E06) are protected against nonalcoholic fatty liver disease (NAFLD). To examine the effect of OxPC elimination at different stages of NAFLD progression, we used cre-dependent, adeno-associated virus serotype 8–mediated expression of the single-chain variable fragment of E06 (AAV8-scFv-E06) in hepatocytes of albumin-cre mice. AAV8-induced expression of scFv-E06 at the start of FPC diet protected mice from developing hepatic steatosis. Independently, expression of scFv-E06 in mice with established steatosis prevented the progression to hepatic fibrosis. Mass spectrometry–based oxophospho-lipidomics identified individual OxPC species that were reduced by scFv-E06 expression. In vitro, identified OxPC species dysregulated mitochondrial metabolism and gene expression in hepatocytes and hepatic stellate cells. We demonstrate that individual OxPC species independently affect disease initiation and progression from hepatic steatosis to steatohepatitis, and that AAV-mediated expression of scFv-E06 is an effective therapeutic intervention.

2022-05-01·The FASEB Journal

Virus‐induced Hepatic Expression of an Oxidized Phospholipid‐binding Antibody Fragment Prevents Initiation of Hepatic Steatosis and Progression to Fibrosis

Article

作者: Manjegowda, Mohan ; Pavelec, Caitlin M. ; Leitinger, Norbert ; Upchurch, Clint M. ; Perez‐Reyes, Edward ; Yeudall, Scott ; Bochkis, Irina M. ; Scott, Michael

Oxidized phospholipids have been implicated in chronic pathologies. It had been shown that hyperlipidemic LDL‐R‐deficient mice transgenic for a single chain fragment of an oxidized phosphatidylcholine (OxPC)‐recognizing IgM E06 (scFv‐E06) were protected from developing non‐alcoholic fatty liver disease (NAFLD). To investigate the role of OxPCs in initiation of diet‐induced hepatic steatosis as well as independently in progression to fibrosis, we used an adeno‐associated virus serotype 8 to induce hepatic expression scFv‐E06 in a cre‐dependent manner (AAV8‐scFv‐E06) in B6. Speer6‐ps1Tg(Alb‐cre)21Mgn/J(Alb‐cre) mice. Virus‐induced expression of scFv‐E06 protected mice from diet‐induced hepatic steatosis by reducing liver triglyceride content and tissue damage as measured by plasma liver enzymes. Separately, expression of scFv‐E06 in mice with established hepatic steatosis prevented the progression to hepatic fibrosis. We developed a targeted, mass spectrometry‐based oxophospholipidomic platform to identify and measure OxPC species that were potentially altered by expression of scFv‐E06. We discovered that several OxPCs in the plasma were significantly reduced by scFv‐E06 expression in both steatosis and fibrosis and identified unique OxPC species that were present in both disease settings. Additionally, we show that the identified OxPC species reduced by scFv‐E06 expression dysregulate gene expression and metabolic function of hepatocytes and hepatic stellate cells in vitro. Together, we show that virus‐induced hepatic expression of scFv‐E06 is sufficient to prevent hepatic steatosis as well as intervene in progression from hepatic steatosis to hepatic fibrosis. Furthermore, we identified unique plasma OxPC species that are reduced by expression of scFv‐E06 and may drive initiation of hepatic steatosis and independently progression to hepatic fibrosis. These data demonstrate an important role for individual OxPCs in NAFLD and may provide clinically relevant biomarkers for diagnosis of hepatic steatosis and fibrosis.

2021-12-14·Circulation1区 · 医学

Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency

1区 · 医学

Article

作者: Liu, Wenli ; Endo-Umeda, Kaori ; Tall, Alan R. ; Xiao, Tong ; Sun, Xiaoli ; Dou, Huijuan ; Yalcinkaya, Mustafa ; Witztum, Joseph L. ; Que, Xuchu ; Fidler, Trevor ; Wang, Nan ; Bick, Alexander ; Kotini, Andriana ; Papapetrou, Eirini P. ; Hardaway, Brian ; Natarajan, Pradeep ; Tsimikas, Sotirios ; Abramowicz, Sandra ; Emdin, Conor ; Olszewska, Malgorzata

Background: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT ) reduces LNK function and that LNK-deficient mice display prominent platelet–neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet–neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. Methods: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr –/– mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2 V617F ). Results:Lnk -deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk –/– platelets promoted increased NETosis when incubated with Lnk –/– neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk –/– platelets, as well as increased priming and response of Lnk –/– neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk –/– mice. We also showed increased NETosis when human induced pluripotent stem cell–derived LNK(TT ) neutrophils were incubated with LNK(TT ) platelet/megakaryocytes, but not in isogenic LNK(CC ) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2 VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk +/– and Jak2 VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2 VF Lnk +/+ controls. Conclusions: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2 VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

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