Monocyte rolling, adhesion, and transmigration across the endothelium are mediated by specific interactions between surface adhesion molecules. This process is fundamental to innate immunity and to ...inflammatory disease, including atherosclerosis, where monocyte egress into the intimal space is central to formation of fatty plaques. Monocytes are a heterogeneous population of three distinct subsets of cells, all of which play different roles in atherosclerosis progression. However, it is not well understood how interactions between different monocyte subsets and the endothelium are regulated. Furthermore, it is appreciated that endothelial adhesion molecules are heavily N-glycosylated, but beyond regulating protein trafficking to the cell surface, whether and if so how these N-glycans contribute to monocyte recruitment is not known. This review discusses how changes in endothelial N-glycosylation may impact vascular and monocytic inflammation. It will also discuss how regulating N-glycoforms on the endothelial surface may allow for the recruitment of specific monocyte subsets to sites of inflammation, and how further understanding in this area may lead to the development of glyco-specific therapeutics in the treatment of cardiovascular disease.
The glycolytic enzyme PKM2 (pyruvate kinase muscle 2) is upregulated in monocytes/macrophages of patients with atherosclerotic coronary artery disease. However, the role of cell type-specific PKM2 in ...the setting of atherosclerosis remains to be defined. We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis.
We generated myeloid cell-specific PKM2
mice on Ldlr (low-density lipoprotein receptor)-deficient background (PKM2
Ldlr
). Controls were littermate PKM2
Ldlr
mice. Susceptibility to atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female mice fed a high-fat Western diet for 14 weeks, starting at 8 weeks.
PKM2 was upregulated in macrophages of Ldlr
mice fed a high-fat Western diet compared with chow diet. Myeloid cell-specific deletion of PKM2 led to a significant reduction in lesions in the whole aorta and aortic sinus despite high cholesterol and triglyceride levels. Furthermore, we found decreased macrophage content in the lesions of myeloid cell-specific PKM2
mice associated with decreased MCP-1 (monocyte chemoattractant protein 1) levels in plasma, reduced transmigration of macrophages in response to MCP-1, and impaired glycolytic rate. Macrophages isolated from myeloid-specific PKM2
mice fed the Western diet exhibited reduced expression of proinflammatory genes, including MCP-1, IL (interleukin)-1β, and IL-12. Myeloid cell-specific PKM2
mice exhibited reduced apoptosis concomitant with enhanced macrophage efferocytosis and upregulation of LRP (LDLR-related protein)-1 in macrophages in vitro and atherosclerotic lesions in vivo. Silencing LRP-1 in PKM2-deficient macrophages restored inflammatory gene expression and reduced efferocytosis. As a therapeutic intervention, inhibiting PKM2 nuclear translocation using a small molecule reduced glycolytic rate, enhanced efferocytosis, and reduced atherosclerosis in Ldlr
mice.
Genetic deletion of PKM2 in myeloid cells or limiting its nuclear translocation reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.
Abstract Integrin α9β1 is known to stabilize leukocyte adhesion to the activated endothelium. We determined the role of myeloid cell α9β1 in early atherosclerosis in two models: α9Mye-KOApoe−/− or ...the Ldlr−/− mice transplanted with bone marrow (BM) from α9Mye-KO mice fed a high-fat “Western” diet for 4 wk. α9Mye-KOApoe−/− mice exhibited reduced early lesions in the aortae and aortic sinuses (P < 0.05 vs α9WT Apoe−/− mice). Similar results were obtained in α9Mye-KO BM→Ldlr−/− mice (P < 0.05 vs α9WT BM→Ldlr−/− mice). Reduced early atherosclerosis in α9Mye-KOApoe−/− mice was associated with decreased neutrophil and neutrophil extracellular traps (NETs) content in the aortic lesions (P < 0.05 vs α9WTApoe−/−). Vascular cell adhesion molecule-1-stimulated neutrophils from α9Mye-KO mice exhibited reduced adhesion, transmigration, and NETs formation (NETosis) (P < 0.05 vs α9WT neutrophils). Reduced NETosis was associated with decreased extracellular signal-regulated kinase phosphorylation, peptidyl arginine deiminase 4, and citrullinated histone H3 expression. In summary, genetic ablation of myeloid cell-specific α9 reduces early atherosclerosis, most likely by reducing neutrophil adhesion, transmigration, and NETosis.
Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an ...important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.
There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that ...nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell–specific PKM2−/− mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe−/−). Controls were littermate PKM2fl/flLysMCre– or PKM2fl/flLysMCre–Apoe−/− mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell–specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet CD41+ deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.
•Nuclear PKM2 is upregulated in neutrophils after the onset of ischemic stroke and promotes neutrophil hyperactivation.•PKM2 deficiency in myeloid cells improves short- and long-term stroke outcome by limiting postischemic cerebral thrombo-inflammation.
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Applying educational theory to medical education research is no different to the way theory drives research and practice in other domains. However, the practical application of theory to research in ...medical education can often be challenging for many different reasons. This article seeks to help researchers by exploring some important questions about educational theory, including what theory is, why it is important, and how it can be used in medical education research.
Applying educational theory to medical education research is no different to the way theory drives research and practice in other domains. However, the practical application of theory to research in medical education can often be challenging for many different reasons. This article seeks to help researchers by exploring some important questions about educational theory, including what theory is, why it is important, and how it can be used in medical education research.
Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants. Clinical studies have linked NEC with antecedent red blood cell (RBC) transfusions, but the ...underlying mechanisms are unclear. Here we report a neonatal murine model to investigate this association. C57BL/6 mouse pups rendered anemic by timed phlebotomy and then given RBC transfusions develop NEC-like intestinal injury with prominent necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon within 12-24 h. The anemic intestine is infiltrated by inflammatory macrophages, which are activated in situ by RBC transfusions via a Toll-like receptor (TLR)-4-mediated mechanism and cause bowel injury. Chelation of RBC degradation products with haptoglobin, absence of TLR4, macrophage depletion, and inhibition of macrophage activation is protective. Intestinal injury worsens with increasing severity and the duration of anemia prior to transfusion, indicating a need for the re-evaluation of current transfusion guidelines for premature infants.
BACKGROUND:Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca signaling in endothelial cells promotes vasodilation has led to the ...hypothesis that endothelial Ca signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels.
METHODS:We used endothelium-specific knockout mice and high-fat diet–fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals.
RESULTS:We show that Ca influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients.
CONCLUSIONS:These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.
Human monocytes have been classified into three distinct groups, classical (anti-inflammatory; CD14
/CD16
), nonclassical (patrolling; CD14
/CD16
), and intermediate (proinflammatory; CD14
/CD16
). ...Adhesion of nonclassical/intermediate monocytes with the endothelium is important for innate immunity, and also vascular inflammatory disease. However, there is an incomplete understanding of the mechanisms that regulate CD16
versus CD16
monocyte adhesion to the inflamed endothelium. Here, we tested the hypothesis that a high-mannose (HM)
-glycoform of intercellular adhesion molecule-1 (ICAM-1) on the endothelium mediates the selective recruitment of CD16
monocytes. Using TNF-α treatment of human umbilical vein endothelial cells (HUVECs), and using proximity ligation assay for detecting proximity of specific
-glycans and ICAM-1, we show that TNF-α induces HM-ICAM-1 formation on the endothelial surface in a time-dependent manner. We next measured CD16
or CD16
monocyte rolling and adhesion to TNF-α-treated HUVECs in which HM- or hybrid ICAM-1
-glycoforms were generated using the α-mannosidase class I and II inhibitors, kifunensine and swainsonine, respectively. Expression of HM-ICAM-1 selectively enhanced CD16
monocyte adhesion under flow with no effect on CD16
monocytes noted. CD16
monocyte adhesion was abrogated by blocking either HM epitopes or ICAM-1. A critical role for HM-ICAM-1 in mediating CD16
monocyte rolling and adhesion was confirmed using COS-1 cells engineered to express HM or complex ICAM-1
-glycoforms. These data suggest that HM-ICAM-1 selectively recruits nonclassical/intermediate CD16
monocytes to the activated endothelium.
Monocyte subsets have been associated with cardiovascular disease, yet it is unknown how different subsets are recruited to the endothelium. This study demonstrates the formation of distinct ICAM-1
-glycoforms in the activated endothelium and reveals a key role for high mannose ICAM-1 in mediating proinflammatory CD16
monocyte adhesion. Presented data identify roles for endothelial
-glycans in recruiting specific monocyte subsets during inflammation.
Purpose
Use of Flory–Huggins interaction parameter and contact angle values to predict the suitability of the drug-polymer system for the production and stability of nanosuspensions.
Material and ...Methods
Melting point depression of the drug was measured using differential scanning calorimetry. Interaction parameter, χ, was calculated using the melting point depression data to elucidate the drug-polymer interaction strength to predict the suitability of the drug-polymer system for the production and stability of nanosuspensions. Contact angle of the drug films were measured with purified water and 0.1%w/w polymer solutions to predict polymer’s suitability for the production and stability of nanosuspension. Nanosuspensions were manufactured to validate the application of the melting point depression approach along with surface property information.
Results
All three polymers, HPMC, Soluplus®, and poloxamer exhibited a negative interaction parameter with naproxen and budesonide. Higher negative interaction parameter values for the naproxen-polymer system indicated stronger drug-polymer interactions, while smaller negative interaction parameter values for the budesonide-polymer system indicated weaker drug-polymer interactions. Interaction parameter was not obtained for fenofibrate with HPMC and Soluplus®, and similarly, no interaction parameter was obtained for carvedilol with HPMC, most likely due to weaker drug-polymer interactions. All three polymers provided lower equilibrium contact angle values when compared to purified water, indicating an affinity for polymers.
Conclusions
Successful production and stability of several nanosuspensions were correlated with Flory–Huggins’s interaction parameter and contact angle values. In the absence of melting point depression, contact angle values can also be used predict the agglomeration tendencies as we have shown for this study.
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