Macrophage-Derived GSDMD Plays an Essential Role in Atherosclerosis and Cross Talk Between Macrophages via the Mitochondria-STING-IRF3/NF-κB Axis.

BACKGROUND: Macrophages play a crucial role in atherosclerotic plaque formation, and the death of macrophages is a vital factor in determining the fate of atherosclerosis. GSDMD (gasdermin D)-mediated pyroptosis is a programmed cell death, characterized by membrane pore formation and inflammatory factor release. METHODS: ApoE-/- and Gsdmd-/- ApoE-/- mice, bone marrow transplantation, and AAV (adeno-associated virus serotype 9)-F4/80-shGSDMD (shRNA-GSDMD) were used to examine the effect of macrophage-derived GSDMD on atherosclerosis. Single-cell RNA sequencing was used to investigate the changing profile of different cellular components and the cellular localization of GSDMD during atherosclerosis. RESULTS: First, we found that GSDMD is activated in human and mouse atherosclerotic plaques and Gsdmd-/- attenuates the atherosclerotic lesion area in high-fat diet-fed ApoE-/- mice. We performed single-cell RNA sequencing of ApoE-/- and Gsdmd-/- ApoE-/- mouse aortas and showed that GSDMD is principally expressed in atherosclerotic macrophages. Using bone marrow transplantation and AAV-F4/80-shGSDMD, we identified the potential role of macrophage-derived GSDMD in aortic pyroptosis and atherosclerotic injuries in vivo. Mechanistically, GSDMD contributes to mitochondrial perforation and mitochondrial DNA leakage and subsequently activates the STING (stimulator of interferon gene)-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) axis. Meanwhile, GSDMD regulates the STING pathway activation and macrophage migration via cytokine secretion. Inhibition of GSDMD with GSDMD-specific inhibitor GI-Y1 (GSDMD inhibitor Y1) can effectively alleviate the progression of atherosclerosis. CONCLUSIONS: Our study has provided a novel macrophage-derived GSDMD mechanism in the promotion of atherosclerosis and demonstrated that GSDMD can be a potential therapeutic target for atherosclerosis.

[Three-dimensional finite element analysis of Swanson prosthesis-arthroplasty of the first metatarsophalangeal joint combined with osteotomy and bone grafting of the first metatarsal bone for hallux valgus].

Objective: To analyze the biomechanical changes of hallux valus after Swanson prosthesis-arthroplasty of the 1st metatarsophalangeal joint combined with osteotomy and bone grafting of the 1st metatarsal bone by three-dimensional finite element analysis, so as to provide data basis for studying the changes of foot morphology and physiological function after hallux valus correction surgery. Methods: A 65-year-old female patient with severe hallux valus admitted in January 2013 was selected as the research object. The CT data of the right foot was obtained, and the three-dimensional finite element models before and after Swanson prosthesis-arthroplasty of the 1st metatarsophalangeal joint combined with osteotomy and bone grafting of the 1st metatarsal bone were established by Mimics10.01, Geomagic Studio, and ANSYS12.0 software. ANSYS 12.0 software was used for nonlinear static stress analysis, and the hallux valgus angle (HVA), the intermetatarsal angle (IMA), and the von Mises stress distributions of the forefoot plantar surface and the 1st to 5th metatarsal bones were observed before and after operation. Results: The HVA and IMA were 56.3° and 16.3° before operation and 9.2° and 9.8° after operation, respectively. Before operation, the stress on the forefoot was the largest in the 4th metatarsal head zone and the smallest in the 1st metatarsal head zone; the stress on the medial side of the forefoot was significantly smaller than that on the lateral side, and the center of forefoot pressure was located on the lateral side. After operation, the stress on the forefoot was the largest in the 1st metatarsal head zone and the smallest in the 5th metatarsal head zone; the stress on the lateral side of the forefoot was significantly smaller than that on the medial side, and the center of forefoot pressure was located on the medial side. Before operation, the stress of the 5th metatarsal bone was the largest, and the 1st metatarsal bone was the smallest. After operation, the stress of the 1st metatarsal bone was the largest, and the 4th metatarsal bone was the smallest. Conclusion: Swanson prosthesis-arthroplasty of the 1st metatarsophalangeal joint combined with osteotomy and bone grafting of the 1st metatarsal bone can effectively correct hallux valgus and make HVA, IMA, and plantar pressure distribution close to normal. However, postoperative stresses of the 1st to 5th metatarsal bones elevate, which may lead to associated complications.

Characterization of fragment sizes, copy number aberrations and 4-mer end motifs in cell-free DNA of hepatocellular carcinoma for enhanced liquid biopsy-based cancer detection.

Circulating cell-free DNA (cfDNA) fragmentomics, which encompasses the measurement of cfDNA length and short nucleotide motifs at the ends of cfDNA molecules, is an emerging field for cancer diagnosis. The utilization of cfDNA fragmentomics for the diagnosis of patients with hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV) is currently limited. In this study, we utilized whole-genome sequencing data of cfDNA in samples from patients with HCC (n = 197) and HBV (n = 187) to analyze the association of fragment size selection (< 150 bp) with tumor fraction (TF), copy number variation (CNV) alterations and the change in the proportion of 4-mer end motifs in HCC and HBV samples. Our analyses identified five typical CNV markers (i.e. loss in chr1p, chr4q and chr8p, and gain in chr1q and chr8q) in cfDNA with a cumulatively positive rate of ˜ 95% in HCC samples. Size selection (< 150 bp) significantly enhanced TF and CNV signals in HCC samples. Additionally, three 4-mer end motifs (CCCA, CCTG and CCAG) were identified as preferred end motifs in HCC samples. We identified 139 end motifs significantly associated with fragment size that showed similar patterns of associations between patients with HCC and HBV, suggesting that end motifs might be inherently coupled with fragment size by a ubiquitous mechanism. Here we conclude that CNV markers, fragment size selection and end-motif pattern in cfDNA have potential for effective detection of patients with HCC.

RBM4 regulates M1 macrophages polarization through targeting STAT1-mediated glycolysis.

M1/M2 macrophages polarization play important roles in regulating tissue homeostasis. Recently, RNA-binding motif 4 (RBM4) has been reported to modulate the proliferation and expression of inflammatory factors in HeLa cells. However, whether RBM4 is involved in regulating macrophage polarization and inflammatory factor expression are still unknown. In this study, RAW264.7, a mouse macrophage cell line, were stimulated with interferon γ (IFN-γ) or interleukin-4 (IL-4) to induce M1/M2 macrophages polarization. We found that IFN-γ, but not IL-4, stimulation decreased RBM4 expression in macrophages, and RBM4 overexpression inhibits IFN-γ-induced M1 macrophage polarization. Furthermore, RNA-Sequencing, protein immunoprecipitation accompanied with mass spectrometry, and extracellular acidification rate analysis showed that RBM4 suppresses IFN-γ-induced M1 macrophage polarization though inhibiting glycolysis. Moreover, RBM4 knockdown promoted IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) activation via increasing STAT1 mRNA stability, leading to the increase of glycolysis-related gene transcripts regulated by STAT1. Finally, we find that RBM4 interacts with YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) to degrade m6A modified STAT1 mRNA, thereby regulating glycolysis and M1 macrophage polarization. Collectively, the current study firstly reports that RBM4 regulates M1 macrophages polarization through targeting STAT1-mediated glycolysis and shows that RBM4 is a possible candidate for regulating macrophage M1 polarization and inflammatory responses.

LncRNA MALAT1 regulates oxLDL-induced CD36 expression via activating ß-catenin.

The expression of scavenger receptors in macrophages regulating lipid uptake plays an important role in foam cell formation and the subsequent atherosclerotic plaque formation. Long non-coding RNA MALAT1 is abundantly expressed in THP-1-derived macrophages, and oxidized low-density lipoprotein promotes its transcription by qRT-PCR and RNA FISH detection. Through chemical inhibitor treatments and by performing a dual luciferase reporter analysis, we found that oxLDL induces MALAT1 transcription through the NF-κB pathway. The knockdown of MALAT1 using siRNA transfection affects lipid uptake in macrophages. To understand the details, we checked the scavenger receptors, which mainly control lipid uptake, and found that MALAT1 knockdown decreased CD36 expression. Additionally, we also incubated macrophages with actinomycin D, combined with a dual luciferase reporter analysis, and we found that MALAT1 influenced CD36 expression at the transcription level. We aim to investigate the detailed mechanism by which MALAT1 promotes CD36 transcription, and thus, we designed and synthesized biotin-TEG labeled oligonucleotides to precipitate the MALAT1 RNA-DNA-protein complex in vivo. Combined with SDS-PAGE electrophoresis and a subsequent mass spectra analysis, ß-catenin, a transcription factor that promotes CD36 transcription, was found in the complex. By performing R-IPs, we validated that ß-catenin was bound to MALAT1 under the oxLDL treatment. In addition, using VAX939, a chemical inhibitor of ß-catenin, MALAT1 was demonstrated to promote CD36 transcription partly via ß-catenin. We also performed chips to detect whether MALAT1 affects ß-catenin accumulation in the binding sites of the CD36 promoter and found that MALAT1 knockdown decreases ß-catenin binding to the CD36 promoter and vice versa. In conclusion, oxLDL induced MALAT1 transcription and MALAT1 recruits ß-catenin to binding sites on the CD36 promoter to induce CD36 expression, which enhances lipid uptake in macrophages.

Association of P2Y12 gene promoter DNA methylation with the risk of clopidogrel resistance in coronary artery disease patients.

BACKGROUND: Clopidogrel inhibits the ADP receptor P2Y12 to keep down the platelet aggregation. The goal of our study is to investigate the contribution of P2Y12 promoter DNA methylation to the risk of clopidogrel resistance (CR). METHODS: The platelet functions were measured by the VerifyNow P2Y12 assay. Applying the bisulfite pyrosequencing technology, DNA methylation levels of two CpG dinucleotides on P2Y12 promoter were tested among 49 CR cases and 57 non-CR controls. We also investigated the association among P2Y12 DNA methylation, various biochemical characteristics, and CR. RESULT: Lower methylation of two CpGs indicated the poorer clopidogrel response (CpG1, P=0.009; CpG2, P=0.022) in alcohol abusing status. Meanwhile CpG1 methylation was inversely correlated with CR in smoking patients (P=0.026) and in subgroup of Albumin<35 (P=0.002). We observed that the level of DNA methylation might be affected by some clinical markers, such as TBIL, LEVF, Albumin, AST. The results also showed that the quantity of stent, fasting blood-glucose, and lower HbAC1 were the predictors of CR. CONCLUSIONS: The evidence from our study indicates that P2Y12 methylation may bring new hints to elaborate the pathogenesis of CR.