Rationale Several reports suggest that antisense oligonucleotides against miR-33 might reduce cardiovascular risk in patients by accelerating the reverse cholesterol transport pathway. However, conflicting reports exist regarding the impact of anti-miR-33 therapy on the levels of very low-density lipoprotein-triglycerides (VLDL-TAG). Objective We test the hypothesis that miR-33 controls hepatic VLDL-TAG secretion. Methods and Results Using therapeutic silencing of miR-33 and adenoviral overexpression of miR-33, we show that miR-33 limits hepatic secretion of VLDL-TAG by targeting N-ethylmaleimide-sensitive factor (NSF), both in vivo and in primary hepatocytes. We identify conserved sequences in the 3’UTR of NSF as miR-33 responsive elements, and show that Nsf is specifically recruited to the RNA-Induced Silencing Complex (RISC) following induction of miR-33. In pulse-chase experiments, either miR-33 overexpression or knock-down of Nsf lead to decreased secretion of apoproteins and TAG in primary hepatocytes, compared to control cells. Importantly, Nsf rescues miR-33–dependent reduced secretion. Finally, we show that overexpression of Nsf in vivo increases global hepatic secretion and raises plasma VLDL-TAG. Conclusion Together, our data reveal key roles for the miR-33–NSF axis during hepatic secretion, and suggest that caution should be taken with anti-miR-33–based therapies since they might raise pro-atherogenic VLDL-TAG levels.

Increased endothelial permeability is central to the pathogenesis of sepsis and leads to organ dysfunction and death but the endogenous mechanisms that drive increased endothelial permeability are not completely understood. We previously reported that cell-free hemoglobin (CFH), elevated in 80% of patients with sepsis, increases lung microvascular permeability in an ex vivo human lung model and cultured endothelial cells. In this study, we augmented a murine model of polymicrobial sepsis with elevated circulating CFH to test the hypothesis that CFH increases microvascular endothelial permeability by inducing endothelial apoptosis. Mice were treated with an intraperitoneal injection of cecal slurry with or without a single intravenous injection of CFH. Severity of illness, mortality, systemic and lung inflammation, endothelial injury and dysfunction and lung apoptosis were measured at selected time points. We found that CFH added to CS increased sepsis mortality, plasma inflammatory cytokines as well as lung apoptosis, edema and inflammation without affecting large vessel reactivity or vascular injury marker concentrations. These results suggest that CFH is an endogenous mediator of increased endothelial permeability and apoptosis in sepsis and may be a promising therapeutic target. OPEN ACCESS Citation: Meegan JE, Shaver CM, Putz ND, Jesse JJ, Landstreet SR, Lee HNR, et al. (2020) Cell-free hemoglobin increases inflammation, lung apoptosis, and microvascular permeability in murine polymicrobial sepsis. PLoS ONE 15(2):

Conflict of interest: LBW reports consulting fees from CSL Behring, Bayer, and Quark Pharmaceuticals as well as a research contract with CSL Behring (current) and past research contracts with Boehringer Ingelheim and Global Blood Therapeutics, all unrelated to the current manuscript. CRP is a named inventor on provisional patent number 62/716,465 titled "System and methods for diagnosing acute interstitial nephritis," which is unrelated to the current manuscript.

Vitamin C (ascorbate, ASC) is a critical antioxidant in the body with specific roles in the brain. Despite a recent interest in vitamin C therapies for critical care medicine, little is known about vitamin C regulation during acute inflammation and critical illnesses such as sepsis. Using a cecal slurry (CS) model of sepsis in mice, we determined ASC and inflammatory changes in the brain following the initial treatment. ASC levels in the brain were acutely decreased by approximately 10% at 4 and 24 h post CS treatment. Changes were accompanied by a robust increase in liver ASC levels of up to 50%, indicating upregulation of synthesis beginning at 4 h and persisting up to 7 days post CS treatment. Several key cytokines interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor alpha (TNFα), and chemokine (C-X-C motif) ligand 1 (CXCL1, KC/Gro) were also significantly elevated in the cortex at 4 h post CS treatment, although these levels returned to normal by 48 h. These data strongly suggest that ASC reserves are directly challenged throughout illness and recovery from sepsis. Given the timescale of this response, decreases in cortical ASC are likely driven by hyper-acute neuroinflammatory processes. However, future studies are required to confirm this relationship and to investigate how this deficiency may subsequently impact neuroinflammation.