Diacylglycerols and Lysophosphatidic Acid, Enriched on Lipoprotein(a), Contribute to Monocyte Inflammation.

BACKGROUND: Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). METHODS: Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. RESULTS: Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1ß after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). CONCLUSIONS: Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.

Enhanced detection rate of Mycoplasma genitalium in urine overtime by transcription-mediated amplification in comparison to real-time PCR.

BACKGROUND: Diagnosis of infected individuals with Mycoplasma genitalium (MG) is often performed by real-time PCR or transcription-mediated amplification (TMA). A limitation of the MG-TMA assay is the relatively short time span of 24 h in which the collected urine is required to be transferred into a Urine Specimen Transport Tube, according to the manufacturer's guidelines. If not transferred within 24 h, the manufacturer's claimed sensitivity cannot be guaranteed anymore, and samples may instead be tested with an in-house validated real-time PCR, despite its recognized lower sensitivity. This study aimed to validate an exception to the sample transport and storage conditions of the MG-TMA assay as set by the manufacturer, being the prolongation of the acceptable testing time limit of 24 h. METHODS: From June to December 2022, first-void urines were collected from clients attending the clinic for sexual health in Amsterdam, the Netherlands. Urine samples that tested positive for MG by TMA assay at the day of collection were concomitantly stored at room (18-24 °C) and refrigerator temperature (4-8 °C) for 15 days. The stored urine samples were tested with both an in-house validated real-time PCR and MG-TMA assay after transfer of the original urine samples to the respective test tubes at 3, 7, 12 and 15 days post collection. RESULTS: In total, 47 MG-positive urine samples were collected, stored and tested for MG by real-time PCR and TMA assays. After storage at room temperature, the MG-detection rate by TMA was significantly higher compared to real-time PCR, at days 0 (p ≤ 0.001), 7 (p ≤ 0.001) and 12 (p < 0.05). After storage at refrigerator temperature, the MG-detection rate determined by TMA assay was significantly enhanced in comparison with real-time PCR at days 3 (p < 0.01), 7 (p ≤ 0.001) and 15 (p < 0.01). CONCLUSIONS: This validation study showed that the MG-TMA assay has a superior detection rate in urine compared to real-time PCR, up to 15 days post sample collection and irrespective of storage temperature. Accepting urines older than 24 h to be tested by TMA will improve clinical diagnosis of MG infections.

Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability.

AIMS: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. METHODS AND RESULTS: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6-8 weeks old male Ldlr -/- mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. CONCLUSION: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis.