Notes

Cancer restorative strategies determined by steel ions.
Familial hypercholesterolemia (FH) is characterized by elevated low-density lipoprotein-cholesterol and markedly increased cardiovascular risk. In T0070907 solubility dmso with a genetic diagnosis, low-density lipoprotein receptor (
) mutations account for >90% of cases, apolipoprotein B (
) mutations for ≈5% of cases, while proprotein convertase subtilisin kexin type 9 (
) gain of function mutations are rare (<1% of cases). We aimed to evaluate the functional impact of several novel
variants in a cohort of patients with FH by genetic cascade screening and in vitro functionality assays. Approach and Results Patients with clinically diagnosed FH underwent genetic analysis of
, and if negative, sequential testing of
and
. We analyzed cosegregation of hypercholesterolemia with novel
variants. Gain of function status was determined by in silico analyses and validated by in vitro functionality assays. Among 1055 persons with clinical FH, we identified nonsynonymous
variants in 27 (2.6%) patients and 7 of these carried one of the 4 previously reported gain of function variants. In the remaining 20 patients with FH, we identified 7 novel
variants. The G516V variant (c.1547G>T) was found in 5 index patients and cascade screening identified 15 additional carriers. Low-density lipoprotein-cholesterol levels were higher in these 15 carriers compared with the 27 noncarriers (236±73 versus 124±35 mg/dL;
0.001). In vitro studies demonstrated the pathogenicity of the G516V variant.

In our study, 1.14% of cases with clinical FH were clearly attributable to pathogenic variants in
. #link# Pathogenicity is established beyond doubt for the G516V variant.
In our study, 1.14% of cases with clinical FH were clearly attributable to pathogenic variants in PCSK9. Pathogenicity is established beyond doubt for the G516V variant.
Septin 2 is localized at junctions in human microvascular endothelial monolayers. The junctional localization of septin 2 is necessary for organization of cell-cell adhesion proteins of endothelial cells. Approach and Results Septin 2 was depleted at junctions by suppression of expression using shRNA, treatment with inflammatory cytokine, TNF (tumor necrosis factor)-α, and ectopic overexpression of septin 2 phosphatidylinositol 4,5-bisphosphate binding mutant defect in interaction with plasma membrane. Under those conditions, organizations and expression levels of various junctional proteins were analyzed. Confocal images of immunofluorescence staining showed substantial disorganization of adherens junctional proteins, nectin-2 and afadin, TJP (tight junction protein), ZO (zonula occludens)-1, and intercellular adhesion protein, PECAM-1 (platelet-endothelial cell adhesion molecule-1). Immunoblots for those proteins did not show significant changes in expression except for nectin-2 that highly increased in expression. Significant differential gene expression profiles and biological pathway analysis by septin 2 suppression and by TNF-α treatment using RNA-seq showed common overlapping pathways. The commonalities in expression may be consistent with the similar effects on the overall organization of cell-cell adhesion proteins.

Localization of septin 2 at cell junctions are required for the arrangement of junctional proteins and the integrity of the barrier formed by endothelial monolayers.
Localization of septin 2 at cell junctions are required for the arrangement of junctional proteins and the integrity of the barrier formed by endothelial monolayers.
The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VIC
) mesenchymal-like phenotype was confirmed by CD90
/CD73
/CD44
expression and multipotent-like differentiation ability. When VIC
were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VIC
and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A d from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.
Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients.

Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.
Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.
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