HAF prevents hepatocyte apoptosis and progression to MASH and hepatocellular carcinoma through transcriptional regulation of the NF-κB pathway.

BACKGROUND: HCC incidence is increasing worldwide due to the obesity epidemic, which drives metabolic dysfunction-associated steatohepatitis (MASH) that can lead to HCC. However, the molecular pathways driving MASH-HCC are poorly understood. We have previously reported that male mice with haploinsufficiency of hypoxia-associated factor, HAF (SART1+/-) spontaneously develop MASH-HCC. However, the cell type(s) responsible for HCC associated with HAF loss are unclear. RESULTS: We generated SART1-floxed mice, which were crossed with mice expressing Cre-recombinase within hepatocytes (Alb-Cre; hepS-/-) or myeloid cells (LysM-Cre, macS-/-). HepS-/- mice (both male and female) developed HCC associated with profound inflammatory and lipid dysregulation suggesting that HAF protects against HCC primarily within hepatocytes. HAF-deficient hepatocytes showed decreased P-p65 and P-p50 and in many components of the NF-κB pathway, which was recapitulated using HAF siRNA in vitro. HAF depletion also triggered apoptosis, suggesting that HAF protects against HCC by suppressing hepatocyte apoptosis. We show that HAF regulates NF-κB activity by regulating transcription of TRADD and RIPK1. Mice fed a high-fat diet (HFD) showed marked suppression of HAF, P-p65 and TRADD within their livers after 26 weeks, but showed profound upregulation of these proteins after 40 weeks, implicating deregulation of the HAF-NF-κB axis in the progression to MASH. In humans, HAF was significantly decreased in livers with simple steatosis but significantly increased in HCC compared with normal liver. CONCLUSIONS: HAF is novel transcriptional regulator of the NF-κB pathway and is a key determinant of cell fate during progression to MASH and MASH-HCC.

ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells.

Magnetic resonance imaging-guided focused ultrasound to increase localized blood-spinal cord barrier permeability.

Spinal cord injury (SCI) affects thousands of people every year in the USA, and most patients are left with some permanent paralysis. Therapeutic options are limited and only modestly affect outcome. To address this issue, we used magnetic resonance imaging-guided focused ultrasound (MRgFUS) as a non-invasive approach to increase permeability in the blood-spinal cord barrier (BSCB). We hypothesize that localized, controlled sonoporation of the BSCB by MRgFUS will aid delivery of therapeutics to the injury. Here, we report our preliminary findings for the ability of MRgFUS to increase BSCB permeability in the thoracic spinal cord of a normal rat model. First, an excised portion of normal rat spinal column was used to characterize the acoustic field and to estimate the insertion losses that could be expected in an MRgFUS blood spinal cord barrier opening. Then, in normal rats, MRgFUS was applied in combination with intravenously administered microbubbles to the spinal cord region. Permeability of the BSCB was indicated as signal enhancement by contrast administered prior to T1-weighted magnetic resonance imaging and verified by Evans blue dye. Neurological testing using the Basso, Beattie, and Breshnahan scale and the ladder walk was normal in 8 of 10 rats tested. Two rats showed minor impairment indicating need for further refinement of parameters. No gross tissue damage was evident by histology. In this study, we have opened successfully the blood spinal cord barrier in the thoracic region of the normal rat spine using magnetic resonance-guided focused ultrasound combined with microbubbles.

Deficiency of phospholipase A2 group 7 decreases intestinal polyposis and colon tumorigenesis in Apc(Min/+) mice.

Platelet-activating factor (PAF) is a naturally occurring phospholipid that mediates diverse effects such as physiological and pathological inflammation, immunosuppression, and cancer. Several lines of evidence support both positive and negative roles for PAF in carcinogenesis. PAF stimulates cell growth, oncogenic transformation, and metastasis, but can also limit proliferation and induce apoptosis. The biological context and microenvironment seem to define whether PAF has pro- or anticarcinogenic effects. To investigate the role of exacerbated PAF signaling in colon cancer, we conducted cell-based and in vivo studies using genetically engineered mice lacking expression of phospholipase A2 group 7 (PLA2G7), an enzyme that specifically metabolizes PAF and structurally related glycerophospholipids. Absence of Pla2g7 robustly decreased intestinal polyposis and colon tumor formation in Apc(Min)(/+) mice, suggesting an antitumorigenic role for PAF in settings characterized by aberrant function of the tumor suppressor Adenomatous polyposis coli (Apc). In colonic epithelial cells, exposure to a PAF analog led to dephosphorylation of Akt at serine-473 and induction of apoptosis. The mechanism of this response involved formation of a complex between ß-arrestin 1 and the Akt phosphatase PHLPP2, and activation of the intrinsic pathway of apoptosis. Our results suggest that strategies based on inhibiting PLA2G7 activity or increasing PAF-mediated signaling hold promise for the treatment of intestinal malignancies that harbor mutations in APC.

Novel mechanism for regulation of plasma platelet-activating factor acetylhydrolase expression in mammalian cells.

The plasma form of PAF-AH [PAF (platelet-activating factor) acetylhydrolase; also known as LpPLA(2) (lipopoprotein-associated phospholipase A(2)), PLA(2)G7] catalyses the release of sn-2 fatty acyl residues from PAF, oxidatively fragmented phospholipids, and esterified isoprostanes. The plasma levels of this enzyme vary widely among mammalian species, including mice and humans, but the mechanisms that account for these differences are largely unknown. We investigated the basis for these variations using molecular and biochemical approaches. We identified an N-terminal domain that played key roles in the determination of steady-state expression levels. The mouse N-terminal domain robustly enhanced protein expression levels, possibly owing to its ability to adopt a globular conformation that is absent in the human protein. We investigated the mechanism(s) whereby the N-terminal stretch modulated PAF-AH levels and found that differential expression was not due to variations in the efficiency of transcription, translation, or mRNA stability. Studies designed to evaluate the ability of precursor forms of PAF-AH to mature to fully active proteins indicated that the N-terminal end of human and mouse PAF-AH played important and opposite roles in this process. These domains also modulated the levels of expression of an unrelated polypeptide by affecting the stability of precursor forms of the protein. These studies provide insights that contribute to our understanding of the molecular features and mechanisms that contribute to differential expression of plasma PAF-AH in mammals.

Mouse plasminogen has oxidized phosphatidylcholine adducts that are not metabolized by lipoprotein-associated phospholipase A2under basal conditions.

We previously showed that plasminogen (Plg) isolated from the plasma of normal human subjects contains 1-2 moles of oxidized phosphatidylcholine (oxPtdPC) adducts/mole of protein. Moreover, we suggested that these species are generated at the hepatic site and speculated that they may play a role in the reported cardiovascular pathogenicity of Plg. We aimed to determine whether mouse Plg also harbors linked oxPtdPCs and whether these molecules are metabolized by lipoprotein-associated phospholipase A(2)/PAF acetylhydrolase (Lp-PLA(2)/PAF-AH), an enzyme specific for hydrolysis of oxPtdPCs. We determined the total concentration of Plg in plasma samples from control (WT) and Lp-PLA(2)-deficient (KO) mice, we isolated Plg, and assessed its content of oxPtdPCs by immunoblot analyses. We also evaluated whether human recombinant Lp-PLA(2) metabolized Plg-linked oxPtdPCs in vivo and in vitro. WT and KO mice expressed comparable levels (14.4-15.8 mg/dL) of plasma Plg, as determined by ELISA. We observed no differences in the content of oxPtdPC in Plg isolated from the two mouse strains and in parallel no changes in oxPtdPC content in mouse Plg following incubation with pure recombinant Lp-PLA(2). Plg from mouse plasma contains oxPtdPC adducts that are not affected by the action of Lp-PLA(2), suggesting that linkage to Plg protects oxPtdPCs from metabolism during their transport in the plasma. This modification may have important physio-pathological implications related to the function of Plg, oxPtdPCs, or both.

Transgenic expression of cyclooxygenase-2 in mouse intestine epithelium is insufficient to initiate tumorigenesis but promotes tumor progression.

We generated mice expressing a COX-2 transgene in colon epithelium and found that they did not develop spontaneous colon tumors. But when treated with azoxymethane, a colon carcinogen, COX-2 mice had a higher tumor load compared to wild-type mice. There was no change in the number of pre-neoplastic lesions, indicating that COX-2 does not affect tumor initiation. Tumors in the COX-2 transgenic mice had higher levels of phosphorylated epidermal growth factor receptor and Akt compared to wild-type mice. Collectively, our data indicate that COX-2 promotes colon tumor progression, but not initiation, and it does so, in part, by activating EGFR and Akt signaling pathways.

Identification of a domain that mediates association of platelet-activating factor acetylhydrolase with high density lipoprotein.

The plasma form of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) inactivates potent lipid messengers such as PAF and modified phospholipids generated in settings of oxidant stress. In humans, PAF-AH circulates in blood in fully active form and associates with high and low density lipoproteins (HDL and LDL). Several studies suggest that the location of PAF-AH affects both the catalytic efficiency and the function of the enzyme in vivo. The distribution of PAF-AH among lipoproteins varies widely among mammals. Here, we report that mouse and human PAF-AHs associate with human HDL particles of different density. We made use of this observation in the development of a binding assay to identify domains required for association of human PAF-AH with human HDL. Sequence comparisons among species combined with domain-swapping and site-directed mutagenesis studies led us to the identification of C-terminal residues necessary for the association of human PAF-AH with human HDL. Interestingly, the region identified is not conserved among PAF-AHs, suggesting that PAF-AH interacts with HDL particles in a manner that is unique to each species. These findings contribute to our understanding of the mechanisms responsible for association of human PAF-AH with HDL and may facilitate future studies aimed at precisely determining the function of PAF-AH in each lipoprotein particle.