On the Structure, Stability, and Cell Uptake of Nanostructured Lipid Carriers for Drug Delivery.

The efficacy of nanostructured lipid carriers (NLC) for drug delivery strongly depends on their stability and cell uptake. Both properties are governed by their compositions and internal structure. To test the effect of the lipid composition of NLC on cell uptake and stability, three kinds of liquid lipids with different degrees of unsaturation are employed. After ensuring homogeneous size distributions, the thermodynamic characteristics, stability, and mixing properties of NLC are characterized. Then the rates and predominant pathways of cell uptake are determined. Although the same surfactant is used in all cases, different uptake rates are observed. This finding contradicts the view that the surface properties of NLC are dominated by the surfactant. Instead, the uptake rates are explained by the structure of the nanocarrier. Depending on the mixing properties, some liquid lipids remain inside the nanocarrier, while other liquid lipids are present on the surface. Nanocarriers with liquid lipids on the surface are taken up more readily by the cells. This shows that the engineering of efficient lipid nanocarriers requires a delicate balance of interactions between all components of the nanocarrier on the molecular level.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

BACKGROUND AND AIMS: Lipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear. APPROACH AND RESULTS: Wild-type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two-photon microscopy, and the findings in Bsep KO mice were compared to common bile duct-ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS-induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule-dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice. CONCLUSIONS: Absence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

PI3K Isoform Signalling in Platelets.

Platelets are unique anucleated blood cells that constantly patrol the vasculature to seal and prevent injuries in a process termed haemostasis. Thereby they rapidly adhere to the subendothelial matrix and recruit further platelets, resulting in platelet aggregates. Apart from their central role in haemostasis, they also kept some of their features inherited by their evolutionary ancestor-the haemocyte, which was also involved in immune defences. Together with leukocytes, platelets fight pathogenic invaders and guide many immune processes. In addition, they rely on several signalling pathways which are also relevant to immune cells. Among these, one of the central signalling hubs is the PI3K pathway. Signalling processes in platelets are unique as they lack a nucleus and therefore transcriptional regulation is absent. As a result, PI3K subclasses fulfil distinct roles in platelets compared to other cells. In contrast to leukocytes, the central PI3K subclass in platelet signalling is PI3K class Iß, which underlines the uniqueness of this cell type and opens new ways for potential platelet-specific pharmacologic inhibition. An overview of platelet function and signalling with emphasis on PI3K subclasses and their respective inhibitors is given in this chapter.

Monoglyceride Lipase Deficiency Is Associated with Altered Thrombogenesis in Mice.

Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl-/-) and platelet-specific Mgl-deficient (platMgl-/-) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mgl-/- mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl-/- mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl-/- mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis.

Hepatectomy-induced apoptotic extracellular vesicles stimulate neutrophils to secrete regenerative growth factors.

BACKGROUND & AIMS: Surgical resection of the cancerous tissue represents one of the few curative treatment options for neoplastic liver disease. Such partial hepatectomy (PHx) induces hepatocyte hyperplasia, which restores liver function. PHx is associated with bacterial translocation, leading to an immediate immune response involving neutrophils and macrophages, which are indispensable for the priming phase of liver regeneration. Additionally, PHx induces longer-lasting intrahepatic apoptosis. Herein, we investigated the effect of apoptotic extracellular vesicles (aEVs) on neutrophil function and their role in this later phase of liver regeneration. METHODS: A total of 124 patients undergoing PHx were included in this study. Blood levels of the apoptosis marker caspase-cleaved cytokeratin-18 (M30) and circulating aEVs were analyzed preoperatively and on the first and fifth postoperative days. Additionally, the in vitro effects of aEVs on the secretome, phenotype and functions of neutrophils were investigated. RESULTS: Circulating aEVs increased at the first postoperative day and were associated with higher concentrations of M30, which was only observed in patients with complete liver recovery. Efferocytosis of aEVs by neutrophils induced an activated phenotype (CD11bhighCD16highCD66bhighCD62Llow); however, classical inflammatory responses such as NETosis, respiratory burst, degranulation, or secretion of pro-inflammatory cytokines were not observed. Instead, efferocytosing neutrophils released various growth factors including fibroblast growth factor-2 and hepatocyte growth factor (HGF). Accordingly, we observed an increase of HGF-positive neutrophils after PHx and a correlation of plasma HGF with M30 levels. CONCLUSIONS: These data suggest that the clearance of PHx-induced aEVs leads to a population of non-inflammatory but regenerative neutrophils, which may support human liver regeneration. LAY SUMMARY: In this study, we show that the surgical removal of a diseased part of the liver triggers a specific type of programmed cell death in the residual liver tissue. This results in the release of vesicles from dying cells into the blood, where they are cleared by circulating immune cells. These respond by secreting hepatocyte growth factors that could potentially support the regeneration of the liver remnant.

More than Just a Monolayer: the Multifaceted Role of Endothelial Cells in the Pathophysiology of Atherosclerosis.

PURPOSE OF THE REVIEW: In this review, we summarize current insights into the versatile roles of endothelial cells in atherogenesis. RECENT FINDINGS: The vascular endothelium represents the first barrier that prevents the entry of lipoproteins and leukocytes into the vessel wall, thereby controlling two key events in the pathogenesis of atherosclerosis. Disturbance of endothelial homeostasis increases vascular permeability, inflammation, and cellular trans-differentiation, which not only promotes the build-up of atherosclerotic plaques but is also involved in life-threatening thromboembolic complications such as plaque rupture and erosion. In this review, we focus on recent findings on endothelial lipoprotein transport, inflammation, cellular transitions, and barrier function. By using cutting-edge technologies such as single-cell sequencing, epigenetics, and cell fate mapping, novel regulatory mechanisms and endothelial cell phenotypes have been discovered, which have not only challenged established concepts of endothelial activation, but have also led to a different view of the disease.

Alternative activation of human macrophages enhances tissue factor expression and production of extracellular vesicles.

Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand if polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte derived macrophages to a proinflammatory and an alternative activation state. Alternative polarization with interleukin-4 and IL-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, also TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription-6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased tissue factor expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset does not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon-γ with an increase in TF production seems to be due to an increase in TF promoter methylation and was reversible when treating these macrophages with a demethylation agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.

Till Death Do Us Part-The Multifaceted Role of Platelets in Liver Diseases.

Platelets are tightly connected with the liver, as both their production and their clearance are mediated by the liver. Platelets, in return, participate in a variety of liver diseases, ranging from non-alcoholic fatty liver diseases, (viral) hepatitis, liver fibrosis and hepatocellular carcinoma to liver regeneration. Due to their versatile functions, which include (1) regulation of hemostasis, (2) fine-tuning of immune responses and (3) release of growth factors and cellular mediators, platelets quickly adapt to environmental changes and modulate disease development, leading to different layers of complexity. Depending on the (patho)physiological context, platelets exert both beneficial and detrimental functions. Understanding the precise mechanisms through which platelet function is regulated at different stages of liver diseases and how platelets interact with various resident and non-resident liver cells helps to draw a clear picture of platelet-related therapeutic interventions. Therefore, this review summarizes the current knowledge on platelets in acute and chronic liver diseases and aims to shed light on how the smallest cells in the circulatory system account for changes in the (patho)physiology of the second largest organ in the human body.

Genetic platelet depletion is superior in platelet transfusion compared to current models.

Genetically modified mice have advanced our knowledge on platelets in hemostasis and beyond tremendously. However, mouse models harbor certain limitations, including availability of platelet specific transgenic strains, and off-target effects on other cell types. Transfusion of genetically modified platelets into thrombocytopenic mice circumvents these problems. Additionally, ex vivo treatment of platelets prior to transfusion eliminates putative side effects on other cell types. Thrombocytopenia is commonly induced by administration of anti-platelet antibodies, which opsonize platelets to cause rapid clearance. However, antibodies do not differentiate between endogenous or exogenous platelets, impeding transfusion efficacy. In contrast, genetic depletion with the inducible diphtheria toxin receptor (iDTR) system induces thrombocytopenia via megakaryocyte ablation without direct effects on circulating platelets. We compared the iDTR system with antibody-based depletion methods regarding their utility in platelet transfusion experiments, outlining advantages and disadvantages of both approaches. Antibodies led to thrombocytopenia within two hours and allowed the dose-dependent adjustment of the platelet count. The iDTR model caused complete thrombocytopenia within four days, which could be sustained for up to 11 days. Neither platelet depletion approach caused platelet activation. Only the iDTR model allowed efficient platelet transfusion by keeping endogenous platelet levels low and maintaining exogenous platelet levels over longer time periods, thus providing clear advantages over antibody-based methods. Transfused platelets were fully functional in vivo, and our model allowed examination of transgenic platelets. Using donor platelets from already available genetically modified mice or ex vivo treated platelets, may decrease the necessity of platelet-specific mouse strains, diminishing off-target effects and thereby reducing animal numbers.

Endothelial dysfunction in adipose triglyceride lipase deficiency.

Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease.

Role of the ubiquitin-proteasome system in cardiac dysfunction of adipose triglyceride lipase-deficient mice.

Systemic deletion of the gene encoding for adipose triglyceride lipase (ATGL) in mice leads to severe cardiac dysfunction due to massive accumulation of neutral lipids in cardiomyocytes. Recently, impaired peroxisome proliferator-activated receptor α (PPARα) signaling has been described to substantially contribute to the observed cardiac phenotype. Disturbances of the ubiquitin-proteasome system (UPS) have been implicated in numerous cardiac diseases including cardiomyopathy, ischemic heart disease, and heart failure. The objective of the present study was to investigate the potential role of UPS in cardiac ATGL deficiency. Our results demonstrate prominent accumulation of ubiquitinated proteins in hearts of ATGL-deficient mice, an effect that was abolished upon cardiomyocyte-directed overexpression of ATGL. In parallel, cardiac protein expression of the ubiquitin-activating enzyme E1a, which catalyzes the first step of the ubiquitination cascade, was significantly upregulated in ATGL-deficient hearts. Dysfunction of the UPS was accompanied by activation of NF-κB signaling. Moreover, the endoplasmic reticulum (ER)-resident chaperon protein disulfide isomerase was significantly upregulated in ATGL-deficient hearts. Chronic treatment of ATGL-deficient mice with the PPARα agonist Wy14,643 improved proteasomal function, prevented NF-κB activation and decreased oxidative stress. In summary, our data point to a hitherto unrecognized link between proteasomal function, PPARα signaling and cardiovascular disease.

Cardiac oxidative stress in a mouse model of neutral lipid storage disease.

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4-5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~2.5-fold upregulation of soluble guanylate cyclase activity and a ~2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.

Platelets induce endoplasmic reticulum stress in macrophages in vitro.

BACKGROUND: Endoplasmic reticulum (ER) stress is a key feature of lipid-laden macrophages and contributes to the development of atherosclerotic plaques. Blood platelets are known to interact with macrophages and fine-tune effector functions such as inflammasome activation and phagocytosis. However, the effect of platelets on ER stress induction is unknown. OBJECTIVES: The objective of this study is to elucidate the potential of platelets in regulating ER stress in macrophages in vitro. METHODS: Bone marrow-derived macrophages and RAW 264.7 cells were incubated with isolated murine platelets, and ER stress and inflammation markers were determined by reverse transcription-quantitative polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. ER morphology was investigated by electron microscopy. Cell viability, lipid accumulation, and activation were measured by flow cytometry. To gain mechanistic insights, coincubation experiments were performed with platelet decoys/releasates as well as lipopolysaccharide, blocking antibodies, and TLR4 inhibitors. RESULTS: Coincubation of platelets and macrophages led to elevated levels of ER stress markers (BIP, IRE1α, CHOP, and XBP1 splicing) in murine and human macrophages, which led to a pronounced enlargement of the ER. Macrophage ER stress was accompanied by increased release of proinflammatory cytokines and intracellular lipid accumulation, but not cell death. Platelet decoys, but not platelet releasates or lysate from other cells, phenocopied the effect of platelets. Blocking TLR4 inhibited inflammatory activation of macrophages but did not affect ER stress induction by platelet coincubation. CONCLUSION: To our knowledge, this study is the first to demonstrate that platelets induce ER stress and unfolded protein response in macrophages by heat-sensitive membrane proteins, independent of inflammatory activation of macrophages.

Effects of Chronic Inflammatory Activation of Murine and Human Arterial Endothelial Cells at Normal Lipoprotein and Cholesterol Levels In Vivo and In Vitro.

The activation of endothelial cells is crucial for immune defense mechanisms but also plays a role in the development of atherosclerosis. We have previously shown that inflammatory stimulation of endothelial cells on top of elevated lipoprotein/cholesterol levels accelerates atherogenesis. The aim of the current study was to investigate how chronic endothelial inflammation changes the aortic transcriptome of mice at normal lipoprotein levels and to compare this to the inflammatory response of isolated endothelial cells in vitro. We applied a mouse model expressing constitutive active IκB kinase 2 (caIKK2)-the key activator of the inflammatory NF-κB pathway-specifically in arterial endothelial cells and analyzed transcriptomic changes in whole aortas, followed by pathway and network analyses. We found an upregulation of cell death and mitochondrial beta-oxidation pathways with a predicted increase in endothelial apoptosis and necrosis and a simultaneous reduction in protein synthesis genes. The highest upregulated gene was ACE2, the SARS-CoV-2 receptor, which is also an important regulator of blood pressure. Analysis of isolated human arterial and venous endothelial cells supported these findings and also revealed a reduction in DNA replication, as well as repair mechanisms, in line with the notion that chronic inflammation contributes to endothelial dysfunction.

R-ketorolac ameliorates cancer-associated cachexia and prolongs survival of tumour-bearing mice.

BACKGROUND: Cancer-associated cachexia (CAC) is a debilitating syndrome associated with poor quality of life and reduced life expectancy of cancer patients. CAC is characterized by unintended body weight reduction due to muscle and adipose tissue loss. A major hallmark of CAC is systemic inflammation. Several non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested for CAC treatment, yet no single medication has proven reliable. R-ketorolac (RK) is the R-enantiomer of a commonly used NSAID. The effect of RK on CAC has not yet been evaluated. METHODS: Ten- to 11-week-old mice were inoculated with C26 or CHX207 cancer cells or vehicle control (phosphate-buffered saline [PBS]). After cachexia onset, 2 mg/kg RK or PBS was administered daily by oral gavage. Body weight, food intake and tumour size were continuously measured. At study endpoints, blood was drawn, mice were sacrificed and tissues were excised. Immune cell abundance was analysed using a Cytek® Aurora spectral flow cytometer. Cyclooxygenase (COX) activity was determined in lung homogenates using a fluorometric kit. Muscle tissues were analysed for mRNA and protein expression by quantitative real-time PCR and western blotting analysis, respectively. Muscle fibre size was determined on histological slides after haematoxylin/eosin staining. RESULTS: Ten-day survival rate of C26-bearing animals was 10% while RK treatment resulted in a 100% survival rate (P = 0.0009). Chemotherapy resulted in a 10% survival rate 14 days after treatment initiation, but all mice survived upon co-medication with RK and cyclophosphamide (P = 0.0001). Increased survival was associated with a protection from body weight loss in C26 (-0.61 ± 1.82 vs. -4.48 ± 2.0 g, P = 0.0004) and CHX207 (-0.49 ± 0.33 vs. -2.49 ± 0.93 g, P = 0.0003) tumour-bearing mice treated with RK, compared with untreated mice. RK ameliorated musculus quadriceps (-1.7 ± 7.1% vs. -27.8 ± 8.3%, P = 0.0007) and gonadal white adipose tissue (-18.8 ± 49% vs. -69 ± 15.6%, P = 0.094) loss in tumour-bearing mice, compared with untreated mice. Mechanistically, RK reduced circulating interleukin-6 (IL-6) concentrations from 334 ± 151 to 164 ± 123 pg/mL (P = 0.047) in C26 and from 93 ± 39 to 35 ± 6 pg/mL (P = 0.0053) in CHX207 tumour-bearing mice. Moreover, RK protected mice from cancer-induced T-lymphopenia (+1.8 ± 42% vs. -49.2 ± 12.1% in treated vs. untreated mice, respectively). RK was ineffective in ameliorating CAC in thymus-deficient nude mice, indicating that the beneficial effect of RK depends on T-cells. CONCLUSIONS: RK improved T-lymphopenia and decreased systemic IL-6 concentrations, resulting in alleviation of cachexia and increased survival of cachexigenic tumour-bearing mice, even under chemotherapy and independent of COX inhibition. Considering its potential, we propose that the use of RK should be investigated in patients suffering from CAC.

NF-κB in monocytes and macrophages - an inflammatory master regulator in multitalented immune cells.

Nuclear factor κB (NF-κB) is a dimeric transcription factor constituted by two of five protein family members. It plays an essential role in inflammation and immunity by regulating the expression of numerous chemokines, cytokines, transcription factors, and regulatory proteins. Since NF-κB is expressed in almost all human cells, it is important to understand its cell type-, tissue-, and stimulus-specific roles as well as its temporal dynamics and disease-specific context. Although NF-κB was discovered more than 35 years ago, many questions are still unanswered, and with the availability of novel technologies such as single-cell sequencing and cell fate-mapping, new fascinating questions arose. In this review, we will summarize current findings on the role of NF-κB in monocytes and macrophages. These innate immune cells show high plasticity and dynamically adjust their effector functions against invading pathogens and environmental cues. Their versatile functions can range from antimicrobial defense and antitumor immune responses to foam cell formation and wound healing. NF-κB is crucial for their activation and balances their phenotypes by finely coordinating transcriptional and epigenomic programs. Thereby, NF-κB is critically involved in inflammasome activation, cytokine release, and cell survival. Macrophage-specific NF-κB activation has far-reaching implications in the development and progression of numerous inflammatory diseases. Moreover, recent findings highlighted the temporal dynamics of myeloid NF-κB activation and underlined the complexity of this inflammatory master regulator. This review will provide an overview of the complex roles of NF-κB in macrophage signal transduction, polarization, inflammasome activation, and cell survival.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr -/- mice.

Background and Aims: Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. Methods: We generated myeloid-specific Stat4 ΔLysM Ldlr -/- , neutrophil-specific Stat4 ΔS100A8 Ldlr -/- , and control Stat4 fl/fl Ldlr -/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat Pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4 ΔLysM Ldlr -/- and Stat4 fl/fl Ldlr -/- bone marrow cells into aged atherosclerotic Apoe -/- mice and detected by flow cytometry. Results: STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in Stat4 ΔLysM Ldlr -/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Conclusions: Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr-/- mice.

Background and aims: Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. Methods: We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. Results: STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Conclusions: Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.