Neutrophil extracellular traps induced by IL-1ß promote endothelial dysfunction and aggravate limb ischemia.

Vascular inflammation and endothelial dysfunction contribute to vascular diseases. While neutrophil extracellular traps (NETs) participate in some vascular pathologies, their roles in lower limb ischemia remain poorly defined. This study investigated the functional significance of NETs in vascular inflammation and remodeling associated with limb ischemia. Single-cell RNA sequencing (scRNA-seq) and flow cytometry revealed neutrophil activation and upregulated NETs formation in human limb ischemia, with immunofluorescence confirming IL-1ß-induced release of NETs for vascular inflammation. Endothelial cell activation was examined via scRNA-seq and western blotting, indicating enhanced proliferation, expression of adhesion molecules (VCAM-1, ICAM-1), inflammatory cytokines (IL-1ß, IL-6) and decreased expression of VE-cadherin, that could be mediated by NETs to exacerbate endothelial inflammation. Mechanistically, NETs altered endothelial cell function via increased pSTAT1/STAT1 signaling. Vascular inflammation and subsequent ischemia were alleviated in vivo by NETosis or IL-1ß inhibition in ischemic mice. IL-1ß-NETs induce endothelial activation and inflammation in limb ischemia by stimulating STAT1 signaling. Targeting NETs may thus represent a novel therapeutic strategy for inflammatory vascular diseases associated with limb ischemia. Graphical abstract of NETs regulation of the development of vascular inflammation in lower limb ischemia via pSTAT1/STAT1 signaling pathway.

Structural and biochemical characterization of active sites mutant in human inorganic pyrophosphatase.

Inorganic pyrophosphatases (PPases) are enzymes that catalyze the conversion of inorganic pyrophosphate (PPi) into phosphate (Pi). Human inorganic pyrophosphatase 1 (Hu-PPase) exhibits high expression levels in a variety of tumors and plays roles in cell proliferation, apoptosis, invasion and metastasis, making it a promising prognostic biomarker and a target for cancer therapy. Despite its widespread presence, the catalytic mechanism of Hu-PPase in humans remains inadequately understood. The signature motif amino acid sequence (DXDPXD) within the active sites of PPases is preserved across different species. In this research, an enzymatic activity assay revealed that mutations led to a notable reduction in enzymatic function, although the impact of the four amino acids on the activity of the pocket varied. To investigate the influence of these residues on the substrate binding and enzymatic function of PPase, the crystal structure of the Hu-PPase-ED quadruple mutant (D116A/D118A/P119A/D121A) was determined at 1.69 Å resolution. The resulting structure maintained a barrel-like shape similar to that of the wild-type, albeit lacking Mg2+ ions. Molecular docking analysis demonstrated a decreased ability of Hu-PPase-ED to bind to PPi. Further, molecular dynamics simulation analysis indicated that the mutation rendered the loop of Mg2+ ion-binding residues less stable. Therefore, the effect on enzyme activity did not result from a change in the gross protein structure but rather from a mutation that abolished the Mg2+-coordinating groups, thereby eliminating Mg2+ binding and leading to the loss of enzyme activity.

Covered Stents for Treatment of Visceral Artery Aneurysms: A Multicenter Study.

PURPOSE: To evaluate the safety and efficacy of covered stents for treatment of visceral artery aneurysms (VAA). METHODS: This multicenter retrospective study included patients with VAAs who were treated with covered stents between January 2015 and December 2020. A total of 65 patients (mean age, 58 years; range, 27-89 years) with 70 VAAs (60 true aneurysms [86%], 10 pseudoaneurysms [14%]) were included. Of the 65, 48 patients (74%) were asymptomatic. Patient demographics, endovascular treatments, and follow-up results were analyzed. RESULTS: All patients received covered stents. The mean diameter was 2.9 cm (range, 1.0-7.6 cm) for symptomatic aneurysms and 2.5 cm (range, 1.0-9.0 cm) for asymptomatic aneurysms. Of the asymptomatic aneurysms, 89% had a saccular shape. The mean distance between the ostium of the artery in which the aneurysm occurred and the aneurysm was 3.9 cm (median, 3.0 cm; range, 0.5-10 cm). Additional coil embolization was used in 7 aneurysms (10%). During the procedure, 68 (97%) aneurysms were completely excluded, while 2 (3%) had a Type Ib endoleak. After a mean follow-up of 20 months (range, 1-75 months), all patients were asymptomatic. Four endoleaks were recorded and left for close observation. Four stents (7%) had mild restenosis, while the rest of the stents were patent. CONCLUSIONS: Placement of covered stents in patients with VAAs excluded aneurysms and maintained artery patency.

A Large Vascular Leiomyoma Arising from the Superficial Femoral Artery: Case Report and Systematic Review.

OBJECTIVE: Vascular leiomyomas are rare begin tumor comprising mature vascular smooth muscles that originate in the tunica media of the blood vessels. Most of the tumors arise from the veins. Only a dozen cases of artery-arising vascular leiomyoma have been reported, most of which are presented as small nodules in the hand. METHODS: Here we report an interesting case of a large artery-arising vascular leiomyoma and perform a systematic review. RESULTS: A 55-year-old man complained a 6 × 3 cm firm, mobile, tender and pulseless mass in the medial region of his left thigh. The computed tomography showed a well-demarcated fusiform tumor encircling the superficial femoral artery and was enhanced significantly with contrast. The patency of the superficial femoral artery was intact. The magnetic resonance image exhibited slightly hypointense relative to skeletal muscle on T1-weighted images and a heterogeneous appearance on T2-weighted images. We performed the en bloc resection of the tumor and used a vascular graft to revascularize the artery through end-to-end anastomosis. After histopathological assessments, the tumor was diagnosed as an artery-arising vascular leiomyoma. We also performed a systematic review on artery-arising leiomyomas, discovering 21 cases. Most of the artery-arising vascular leiomyomas were small nodules (mean length: 2.4cm) and most of them were superficial solitary mass located in the hand (13 cases, 62%). Excision of the tumor was an effective treatment. The histological subtype of the artery-arising vascular leiomyoma in all cases was solid type. During the follow-up of each patient, there was no recurrence. CONCLUSIONS: Artery-arising vascular leiomyomas are extremely rare. Most of them are painless and locate in the hand. Their pathological subtype is solid type in all patients. Due to their begin nature, excision is a cure with little chance of recurrence.

Uncommon Peptide Bond Cleavage of Glucagon from a Specific Vendor under near Neutral to Basic Conditions.

PURPOSE: The main purposes of this manuscript are to report a surprising and interesting degradation reaction of glucagon from a specific vendor in which glucagon underwent cleavage among several peptide bonds quickly under near neutral to basic conditions, and to propose the root cause of mechanism for the degradation reaction. METHODS: The degradation reaction was monitored by HPLC and the fragment structures were confirmed by LC-MS. Possible impurities responsible for the degradation were either confirmed or excluded by a variety of techniques such as addition of chelator EDTA and transitional metal ions or separation by ultrafiltration. RESULTS: This type of degradation was rarely reported in literature, especially considering its extreme cleavage efficiency. Contamination by a thermostable high molecular impurity (such as a peptidase with molecular weight between 10 and 30 KDa) during the manufacturing process was the main reason for this interesting phenomenon. CONCLUSIONS: The degradation phenomenon described here could be used as an excellent example showing that products ordered from vendors meeting the rudimentary quality standards might contain impurities which could cause significant degradation. We suggest that a simple solution, i.e. additional tests of stability under real or accelerated conditions by manufacturers and inclusion of the "accelerated stability criteria" in the Certificate of Analysis (CoAs), especially for sensitive biological reagents prone to faster degradation, would be very helpful for avoiding losses for both vendors and users.