Inhibition of Neutral Sphingomyelinase 2 by Novel Small Molecule Inhibitors Results in Decreased Release of Extracellular Vesicles by Vascular Smooth Muscle Cells and Attenuated Calcification.

Vascular calcification (VC) is an important contributor and prognostic factor in the pathogenesis of cardiovascular diseases. VC is an active process mediated by the release of extracellular vesicles by vascular smooth muscle cells (VSMCs), and the enzyme neutral sphingomyelinase 2 (nSMase2 or SMPD3) plays a key role. Upon activation, the enzyme catalyzes the hydrolysis of sphingomyelin, thereby generating ceramide and phosphocholine. This conversion mediates the release of exosomes, a type of extracellular vesicles (EVs), which ultimately forms the nidus for VC. nSMase2 therefore represents a drug target, the inhibition of which is thought to prevent or halt VC progression. In search of novel druglike small molecule inhibitors of nSMase2, we have used virtual ligand screening to identify potential ligands. From an in-silico collection of 48,6844 small druglike molecules, we selected 996 compounds after application of an in-house multi-step procedure combining different filtering and docking procedures. Selected compounds were functionally tested in vitro; from this, we identified 52 individual hit molecules that inhibited nSMase2 activity by more than 20% at a concentration of 150 µM. Further analysis showed that five compounds presented with IC50s lower than 2 µM. Of these, compounds ID 5728450 and ID 4011505 decreased human primary VSMC EV release and calcification in vitro. The hit molecules identified here represent new classes of nSMase2 inhibitors that may be developed into lead molecules for the therapeutic or prophylactic treatment of VC.

Reduced Vitamin K Status as a Potentially Modifiable Risk Factor of Severe Coronavirus Disease 2019.

BACKGROUND: Respiratory failure and thromboembolism are frequent in severe acute respiratory syndrome coronavirus 2-infected patients. Vitamin K activates both hepatic coagulation factors and extrahepatic endothelial anticoagulant protein S, required for thrombosis prevention. In times of vitamin K insufficiency, hepatic procoagulant factors are preferentially activated over extrahepatic proteins. Vitamin K also activates matrix Gla protein (MGP), which protects against pulmonary and vascular elastic fiber damage. We hypothesized that vitamin K may be implicated in coronavirus disease 2019 (COVID-19), linking pulmonary and thromboembolic disease. METHODS: A total of 135 hospitalized COVID-19 patients were compared with 184 historic controls. Inactive vitamin K-dependent MGP (desphospho-uncarboxylated [dp-uc] MGP) and prothrombin (PIVKA-II) were measured inversely related to extrahepatic and hepatic vitamin K status, respectively. Desmosine was measured to quantify the rate of elastic fiber degradation. Arterial calcification severity was assessed using computed tomography. RESULTS: dp-ucMGP was elevated in COVID-19 patients compared with controls (P < .001), with even higher dp-ucMGP in patients with poor outcomes (P < .001). PIVKA-II was normal in 82.1% of patients. dp-ucMGP was correlated with desmosine (P < .001) and with coronary artery (P = .002) and thoracic aortic (P < .001) calcification scores. CONCLUSIONS: dp-ucMGP was severely increased in COVID-19 patients, indicating extrahepatic vitamin K insufficiency, which was related to poor outcome; hepatic procoagulant factor II remained unaffected. These data suggest pneumonia-induced extrahepatic vitamin K depletion leading to accelerated elastic fiber damage and thrombosis in severe COVID-19 due to impaired activation of MGP and endothelial protein S, respectively.

Association of Red Blood Cell Distribution Width and Neutrophil-to-Lymphocyte Ratio with Calcification and Cardiovascular Markers in Chronic Kidney Disease.

We aimed to investigate the association between Red Blood Cell Distribution Width (RDW) and Neutrophil-to-Lymphocyte Ratio (NLR), simple, rapidly assessed markers from the complete blood count with vascular calcification (VC)/stiffness and cardiovascular disease (CVD) in chronic kidney disease (CKD). Dephosphorylated, uncarboxylated matrix Gla-protein (dp-ucMGP), and central/peripheral hemodynamics' parameters were measured in 158 CKD patients, including Hemodialysis and Peritoneal Dialysis. Spearman's rho analysis showed that RDW correlated with C-reactive protein (CRP) (r = 0.29, p < 0.001), dp-ucMGP (r = 0.43, p = < 0.0001), central diastolic blood pressure (DBP) (r = -0.19, p = 0.02), and albuminuria (r = -0.17, p = 0.03). NLR correlated with the duration of CVD (r = 0.32, p < 0.001), CRP (r = 0.27, p = 0.01), dp-ucMGP (r = 0.43, p < 0.0001), central DBP (r = -0.32, p < 0.0001) and eGFR (r = -0.25, p = 0.04). In multiple regression models, circulating dp-ucMGP was an independent predictor of RDW (ß = 0.001, p = 0.001) and NLR (ß = 0.002, p = 0.002). In CKD patients, RDW and NLR are associated with traditional and novel markers of VC and CVD.

Exclusively breastmilk-fed preterm infants are at high risk of developing subclinical vitamin K deficiency despite intramuscular prophylaxis at birth.

BACKGROUND: There is near-global consensus that all newborns be given parenteral vitamin K1 (VK1 ) at birth as prophylaxis against VK deficiency bleeding (VKDB). Breastmilk has a low VK content and cases of late VKDB are reported in exclusively breastmilk-fed preterm infants despite VK prophylaxis at birth. OBJECTIVES: To assess the prevalence of functional VK insufficiency in preterm infants based on elevated under-γ-carboxylated (Glu) species of Gla proteins, factor II (PIVKA-II), and osteocalcin (GluOC), synthesized by liver and bone, respectively. PATIENTS/METHODS: Prospective, multicenter, observational study in preterm infants born <33 weeks' gestation. Blood samples and dietary history were collected before hospital discharge, and after discharge at 2-3 months' corrected age. Outcome measures were serum VK1 , PIVKA-II, and %GluOC (GluOC as a percentage of the sum of GluOC plus GlaOC) compared between exclusively breastmilk-fed and formula/mixed-fed infants after discharge. RESULTS: After discharge, breastmilk-fed babies had significantly lower serum VK1 (0.15 vs. 1.81 µg/L), higher PIVKA-II (0.10 vs. 0.02 AU/ml) and higher %GluOC (63.6% vs. 8.1%) than those receiving a formula/mixed-feed diet. Pre-discharge (based on elevated PIVKA-II), only one (2%) of 45 breastmilk-fed infants was VK insufficient. After discharge, eight (67%) of 12 exclusively breastmilk-fed babies were VK insufficient versus only one (4%) of 25 formula/mixed-fed babies. CONCLUSIONS: Preterm infants who remain exclusively or predominantly human breastmilk-fed after neonatal unit discharge are at high risk of developing subclinical VK deficiency in early infancy. Routine postdischarge VK1 supplementation of breastfed infants to provide intakes comparable to those from formula milks should prevent this deficiency.

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions.

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.

A Novel Biomimetic Tool for Assessing Vitamin K Status Based on Molecularly Imprinted Polymers.

Vitamin K was originally discovered as a cofactor required to activate clotting factors and has recently been shown to play a key role in the regulation of soft tissue calcification. This property of vitamin K has led to an increased interest in novel methods for accurate vitamin K detection. Molecularly Imprinted Polymers (MIPs) could offer a solution, as they have been used as synthetic receptors in a large variety of biomimetic sensors for the detection of similar molecules over the past few decades, because of their robust nature and remarkable selectivity. In this article, the authors introduce a novel imprinting approach to create a MIP that is able to selectively rebind vitamin K1. As the native structure of the vitamin does not allow for imprinting, an alternative imprinting strategy was developed, using the synthetic compound menadione (vitamin K3) as a template. Target rebinding was analyzed by means of UV-visible (UV-VIS) spectroscopy and two custom-made thermal readout techniques. This analysis reveals that the MIP-based sensor reacts to an increasing concentration of both menadione and vitamin K1. The Limit of Detection (LoD) for both compounds was established at 700 nM for the Heat Transfer Method (HTM), while the optimized readout approach, Thermal Wave Transport Analysis (TWTA), displayed an increased sensitivity with a LoD of 200 nM. The sensor seems to react to a lesser extent to Vitamin E, the analogue under study. To further demonstrate its potential application in biochemical research, the sensor was used to measure the absorption of vitamin K in blood serum after taking vitamin K supplements. By employing a gradual enrichment strategy, the sensor was able to detect the difference between baseline and peak absorption samples and was able to quantify the vitamin K concentration in good agreement with a validation experiment using High-Performance Liquid Chromatography (HPLC). In this way, the authors provide a first proof of principle for a low-cost, straightforward, and label-free vitamin K sensor.

Annexin A5 inhibits engulfment through internalization of PS-expressing cell membrane patches.

Apoptosis and subsequent clearance of apoptotic cells are important for the prevention of diseases. Therefore, it is essential to understand the mechanisms underlying the biology of phagocytic clearance of apoptotic cells. The best characterized "eat me" signal on the surface of apoptotic cells is phosphatidylserine (PS). Recently, we demonstrated that annexin A5 mediates the internalization of PS-expressing membrane patches and down regulates surface expression of tissue factor. Here, we investigated the role of PS in the phagocytosis of apoptotic cells using annexin A5. Using a novel flow cytometric-based phagocytosis assay, we observed that engulfment was inhibited with 20% if annexin A5 was added to PS-expressing cells that had completed apoptosis. The inhibition increased to more than 50% if annexin A5 was added during the apoptotic process. This inhibition is specific for annexin A5, since the mutant M23 and annexin A1 did not further increase the inhibition of phagocytosis when added during the apoptotic process. Interestingly, cells with internalized annexin A5 still express PS at their surface. We conclude that other ligands within the PS-expressing membrane patch act together with PS as an "eat me" signal.

In vitro measurement of cell death with the annexin A5 affinity assay.

One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.