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Background: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering., Methods: Smooth muscle cell lineage-tracing Apoe -/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1β or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis., Results: Lipid lowering by switching Apoe -/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1β antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1β antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap., Conclusions: Taken together, IL-1β appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability., Competing Interests: Disclosures None.

Neutrophil elastase (NE) is taken up by macrophages, retains intracellular protease activity, and induces a pro-inflammatory phenotype. However, the mechanism of NE-induced pro-inflammatory polarization of macrophages is not well understood. We hypothesized that intracellular NE degrades histone deacetylases (HDAC) and Sirtuins, disrupting the balance of lysine acetylation and deacetylation and resulting in nuclear to cytoplasmic translocation of a major alarmin, High Mobility Group Box 1 (HMGB1), a pro-inflammatory response in macrophages. Human blood monocytes were obtained from healthy donors or from subjects with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Monocytes were differentiated into blood monocyte derived macrophages (BMDMs) in vitro. Human BMDMs were exposed to NE or control vehicle, and the abundance of HDACs and Sirtuins was determined by Western blotting of total cell lysates or nuclear extracts or determined by ELISA. HDAC, Sirtuin, and Histone acetyltransferase (HAT) activities were measured. NE degraded most HDACs and Sirtuin (Sirt)1, resulting in decreased HDAC and sirtuin activities, with minimal change in HAT activity. We then evaluated whether the NE-induced loss of Sirt activity or loss of HDAC activities would alter the cellular localization of HMGB1. NE treatment or treatment with Trichostatin A (TSA), a global HDAC inhibitor, both increased HMGB1 translocation from the nucleus to the cytoplasm, consistent with HMGB1 activation. NE significantly degraded Class I and II HDAC family members and Sirt 1, which shifted BMDMs to a pro-inflammatory phenotype.

Neutrophil elastase (NE), a major inflammatory mediator in chronic obstructive pulmonary disease (COPD) airways, impairs macrophage function, contributing to persistence of airway inflammation. We hypothesized that NE activates a novel mechanism of macrophage-induced inflammation: release of macrophage extracellular traps (METs). The METs are composed of extracellular DNA decorated with granule proteinases and oxidants and may trigger persistent airway inflammation in COPD. To test the hypothesis, human blood monocytes were isolated from whole blood of subjects with COPD recruited following informed written consent. Patient demographics and clinical data were collected. Cells were cultured in media with GM-CSF to differentiate into blood monocyte derived macrophages (BMDMs). The BMDMs were treated with FITC-NE and unlabeled NE to determine intracellular localization by confocal microscopy and intracellular proteinase activity by DQ-Elastin assay. After NE exposure, released extracellular traps were quantified by abundance of extracellular DNA in conditioned media using the Pico Green assay. BMDM cell lysates were analyzed by Western analysis for proteolytic degradation of histone H3 or H4 or upregulation of peptidyl arginine deiminase (PAD) 2 and 4, two potential mechanisms to mediate extracellular trap DNA release. We observed that NE was taken up by COPD BMDM, localized to the cytosol and nucleus, and retained proteinase activity in the cell. NE induced MET release at doses as low as 50 nM. NE treatment caused histone H3 clipping but no effect on histone H4 nor PAD 2 or 4 abundance or activity. In summary, NE activated COPD MET release by clipping histone H3, a prerequisite for chromatin decondensation., (© 2023 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Aims: Until recently, the pluripotency factor Octamer (ATGCAAAT)-binding transcriptional factor 4 (OCT4) was believed to be dispensable in adult somatic cells. However, our recent studies provided clear evidence that OCT4 has a critical atheroprotective role in smooth muscle cells. Here, we asked if OCT4 might play a functional role in regulating endothelial cell (EC) phenotypic modulations in atherosclerosis., Methods and Results: Specifically, we show that EC-specific Oct4 knockout resulted in increased lipid, LGALS3+ cell accumulation, and altered plaque characteristics consistent with decreased plaque stability. A combination of single-cell RNA sequencing and EC-lineage-tracing studies revealed increased EC activation, endothelial-to-mesenchymal transitions, plaque neovascularization, and mitochondrial dysfunction in the absence of OCT4. Furthermore, we show that the adenosine triphosphate (ATP) transporter, ATP-binding cassette (ABC) transporter G2 (ABCG2), is a direct target of OCT4 in EC and establish for the first time that the OCT4/ABCG2 axis maintains EC metabolic homeostasis by regulating intracellular heme accumulation and related reactive oxygen species production, which, in turn, contributes to atherogenesis., Conclusions: These results provide the first direct evidence that OCT4 has a protective metabolic function in EC and identifies vascular OCT4 and its signalling axis as a potential target for novel therapeutics., Competing Interests: Conflict of interest: none declared., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2022. For permissions, please email: journals.permissions@oup.com.)

Caspase 9 undergoes alternative splicing to produce two opposing isoforms: proapoptotic Caspase 9a and pro-survival Caspase 9b (C9b). Previously, our laboratory reported that C9b is expressed in majority of non-small cell lung cancer tumors and directly activates the NF-κB pathway. In this study, the role of C9b in activation of the NF-κB pathway in vivo, lung inflammation and immune responses, and lung tumorigenesis were examined. Specifically, a transgenic mouse model expressing human C9b in the lung pneumocytes developed inflammatory lung lesions, which correlated with enhanced activation of the NF-κB pathway and increased influx of immunosuppressive myeloid-derived suppressor cells in contrast to wild-type mice. C9b mice presented with facial dermatitis, a thickened and disorganized dermis, enhanced collagen depth, and increased serum levels of IL6. C9b mice also developed spontaneous lung tumors, and C9b cooperated with oncogenic KRAS in lung tumorigenesis. C9b expression also cooperated with oncogenic KRAS and p53 downregulation to drive the full cell transformation of human bronchial epithelial cells (e.g., tumor formation)., Implications: Our findings show that C9b can directly activate NF-κB pathway in vivo to modulate lung inflammation, immune cell influx, and peripheral immune responses, which demonstrates that C9b is key factor in driving cell transformation and lung tumorigenesis., (©2022 The Authors; Published by the American Association for Cancer Research.)

Objective: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4 -dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-Cre ERT2 eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP + cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP + ; and unintended proinflammatory consequences. Using newly generated Myh11-Dre ERT2 tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4 Δ/Δ ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1 + lymphatic endothelial cells in the epididymal AT., Conclusions: Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

Objectives: To investigate the color relationships between the anterior teeth in vivo., Materials and Methods: A total of 640 volunteers (age: 18-22) participated in the study. The color measurements of left maxillary and mandibular central, lateral, and canine were performed using a colorimeter. Color differences were calculated according to the CIE L*a*b* (ΔE*) and CIEDE2000 (ΔE 00 *) system. The National Bureau of Standards (NBS) ratings were also evaluated. Statistical analyses were performed using two-way ANOVA and Fisher's exact test (P < 0.05)., Results: Significant differences were found between the L*, a*, b*, and ΔE* values (P < 0.05), except for mandibular central and lateral. ΔE* values were found between 1.5 ΔE* (mandibular central-mandibular lateral) and 8.1 ΔE* (maxillary central-maxillary canine). While the highest L* (80.5) and the least b* (15.1) values were obtained for mandibular central, the mean L* (73.6) and the mean b* values (21.3) were obtained for the maxillary canine. a* value was found to be highest for the mandibular lateral (1.1) and the least for the maxillary central (-0.2) teeth. NBS values were between 1.3 and 7.4 units, and only mandibular central and mandibular lateral teeth exhibited "almost the same" color values, while the other teeth exhibited "slightly different," "very different," or "remarkably different" color values., Conclusion: Anterior maxillary and mandibular teeth exhibited "different" color values. Only mandibular incisors and mandibular laterals showed "almost the same" color. Canines were more dark/red/yellow for both mandible and maxilla. Centrals were more bright/green/yellow for maxilla and bright/green/blue for mandibular. Clinicians should select the color one by one for each tooth and be aware of their color relations for optimal esthetics., Competing Interests: There are no conflicts of interest

Purpose: To assess the fracture resistance of single-tooth implant-supported crown restorations made with different CAD/CAM blocks., Materials and Methods: Thirty-six titanium abutments were put on dental implant analogs (Mis Implant). For each of three test groups (n = 12/group), implant-supported, cement-retained mandibular molar single crowns were produced. Crowns were made of lithium disilicate glass (LD) IPS e.max CAD, feldspathic glass ceramic (FEL) Vita Mark II, and resin nano-ceramic (RNC) Lava Ultimate. The crowns were cemented with self-adhesive resin cement RelyX Unicem 2. After chewing cycling, crowns were tested to failure in a universal testing machine. Fracture values were calculated as initial (F-initial) and maximum fracture (F-max)., Results: The study groups were ranked, in order of having highest value, (LD > FEL) > RNC for F-initial load value and (LD > RNC) > FEL for F-max load value. This demonstrated that there was no parallel change in the F-initial and F-max values presenting the fracture resistance of specimens., Conclusions: There was no accordance between the F-initial and F-max values of the LD, RNC, and FEL after chewing simulation with thermocycling resembling 5 years of clinical functional use. LD had the highest fracture resistance during the fracture test. RNC had low fracture resistance; however, it had considerably high fracture resistance during the fracture test. FEL had considerably low fracture resistance values., (© 2015 by the American College of Prosthodontists.)

Alternate RNA processing of caspase-9 generates the splice variants caspase 9a (C9a) and caspase 9b (C9b). C9b lacks a domain present in C9a, revealing a tumorigenic function that drives the phenotype of non-small cell lung cancer (NSCLC) cells. In this study, we elucidated the mechanistic underpinnings of the malignant character of this splice isoform. In NSCLC cells, C9b expression correlated with activation of the canonical arm of the NF-κB pathway, a major pathway linked to the NSCLC tumorigenesis. Mechanistic investigations revealed that C9b activates this pathway via direct interaction with cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b, but did not require proteolytic cleavage of C9b. This protein:protein interaction was essential for C9b to promote viability and malignant growth of NSCLC cells in vitro and in vivo, broadly translating to diverse NSCLC oncogenotypes. Overall, our findings identified a novel point for therapeutic invention in NSCLC that may be tractable to small-molecule inhibitors, as a new point to broadly address this widespread deadly disease. Cancer Res; 76(10); 2977-89. ©2016 AACR., (©2016 American Association for Cancer Research.)

Nitrogen permease regulator-like 2 (NPRL2) is a component of a conserved complex that inhibits mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid insufficiency. Here, we show that NPRL2 is required for mouse viability and that its absence significantly compromises fetal liver hematopoiesis in developing embryos. Moreover, NPRL2 KO embryos have significantly reduced methionine levels and exhibit phenotypes reminiscent of cobalamin (vitamin B12) deficiency. Consistent with this idea, NPRL2 KO liver and mouse embryonic fibroblasts (MEFs) show defective processing of the cobalamin-transport protein transcobalamin 2, along with impaired lysosomal acidification and lysosomal gene expression. NPRL2 KO MEFs exhibit a significant defect in the cobalamin-dependent synthesis of methionine from homocysteine, which can be rescued by supplementation with cyanocobalamin. Taken together, these findings demonstrate a role for NPRL2 and mTORC1 in the regulation of lysosomal-dependent cobalamin processing, methionine synthesis, and maintenance of cellular re-methylation potential, which are important during hematopoiesis., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Erythropoietin (Epo) binding to the Epo receptor (EpoR) elicits downstream signaling that is essential for red blood cell production. One important negative regulatory mechanism to terminate Epo signaling is Epo-induced EpoR endocytosis and degradation. Defects in this mechanism play a key role in the overproduction of erythrocytes in primary familial and congenital polycythemia (PFCP). Here we have identified a novel mechanism mediating Epo-dependent EpoR internalization. Epo induces Cbl-dependent ubiquitination of the p85 regulatory subunit of PI3K, which binds to phosphotyrosines on EpoR. Ubiquitination allows p85 to interact with the endocytic protein epsin-1, thereby driving EpoR endocytosis. Knockdown of Cbl, expression of its dominant negative forms, or expression of an epsin-1 mutant devoid of ubiquitin-interacting motifs all compromise Epo-induced EpoR internalization. Mutated EpoRs mimicking those from PFCP patients cannot bind p85, co-localize with epsin-1, or internalize on Epo stimulation and exhibit Epo hypersensitivity. Similarly, knockdown of Cbl also causes Epo hypersensitivity in primary erythroid progenitors. Restoring p85 binding to PFCP receptors rescues Epo-induced epsin-1 co-localization and EpoR internalization and normalizes Epo hypersensitivity. Our results uncover a novel Cbl/p85/epsin-1 pathway in EpoR endocytosis and show that defects in this pathway contribute to excessive Epo signaling and erythroid hyperproliferation in PFCP.

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA (miRNA) libraries. With this strategy, we matched proteins and miRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected siRNAs, miRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets.

Ubiquitination is a common mechanism of down-regulation of mitogenic receptors. Here, we show that ubiquitination of the erythropoietin receptor (EpoR) at Lys(256) is necessary and sufficient for efficient Epo-induced receptor internalization, whereas ubiquitination at Lys(428) promotes trafficking of activated receptors to the lysosomes for degradation. Interestingly, EpoR that cannot be ubiquitinated has reduced mitogenic activities and ability to stimulate the STAT5, Ras/MAPK, and PI3K/AKT signaling pathways. We therefore propose that ubiquitination of the EpoR critically controls both receptor down-regulation and downstream signaling.

Primary familial and congenital polycythemia (PFCP) is an autosomal-dominant proliferative disorder characterized by erythrocytosis and hypersensitivity of erythroid progenitors to erythropoietin (Epo). Several lines of evidence suggest a causal role of truncated erythropoietin receptor (EpoR) in this disease. In this review, we discuss PFCP in the context of erythrocytosis and EpoR signalling. We focus on recent studies describing mechanisms underlying Epo-dependent EpoR down-regulation. One mechanism depends on internalization mediated through the p85 regulatory subunit of the Phosphoinositide 3-Kinase, and the other utilizes ubiquitin-based proteasomal degradation. Truncated PFCP EpoRs are not properly down-regulated upon stimulation, underscoring the importance of these mechanisms in the pathogenesis of PFCP.