PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke.

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.

Myeloid Cell PKM2 Deletion Enhances Efferocytosis and Reduces Atherosclerosis.

BACKGROUND: 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. METHODS: We generated myeloid cell-specific PKM2-/- mice on Ldlr (low-density lipoprotein receptor)-deficient background (PKM2mye-KOLdlr-/-). Controls were littermate PKM2WTLdlr-/- 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. RESULTS: 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)-1ß, 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. CONCLUSIONS: Genetic deletion of PKM2 in myeloid cells or limiting its nuclear translocation reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.

Targeting Neutrophil α9 Improves Functional Outcomes After Stroke in Mice With Obesity-Induced Hyperglycemia.

BACKGROUND: Obesity-induced hyperglycemia is a significant risk factor for stroke. Integrin α9ß1 is expressed on neutrophils and stabilizes adhesion to the endothelium via ligands, including Fn-EDA (fibronectin containing extra domain A) and tenascin C. Although myeloid deletion of α9 reduces susceptibility to ischemic stroke, it is unclear whether this is mediated by neutrophil-derived α9. We determined the role of neutrophil-specific α9 in stroke outcomes in a mice model with obesity-induced hyperglycemia. METHODS: α9Neu-KO (α9fl/flMRP8Cre+) and littermate control α9WT (α9fl/flMRP8Cre-) mice were fed on a 60% high-fat diet for 20 weeks to induce obesity-induced hyperglycemia. Functional outcomes were evaluated up to 28 days after stroke onset in mice of both sexes using a transient (30 minutes) middle cerebral artery ischemia. Infarct volume (magnetic resonance imaging) and postreperfusion thrombo-inflammation (thrombi, fibrin, neutrophil, phospho-nuclear factor kappa B [p-NFκB], TNF [tumor necrosis factor]-α, and IL [interleukin]-1ß levels, markers of neutrophil extracellular traps) were measured post 6 or 48 hours of reperfusion. In addition, functional outcomes (modified Neurological Severity Score, rota-rod, corner, and wire-hanging test) were measured for up to 4 weeks. RESULTS: Stroke upregulated neutrophil α9 expression more in obese mice (P<0.05 versus lean mice). Irrespective of sex, deletion of neutrophil α9 improved functional outcomes up to 4 weeks, concomitant with reduced infarct, improved cerebral blood flow, decreased postreperfusion thrombo-inflammation, and neutrophil extracellular traps formation (NETosis) (P<0.05 versus α9WT obese mice). Obese α9Neu-KO mice were less susceptible to thrombosis in FeCl3 injury-induced carotid thrombosis model. Mechanistically, we found that α9/cellular fibronectin axis contributes to NETosis via ERK (extracellular signal-regulated kinase) and PAD4 (peptidyl arginine deiminase 4), and neutrophil α9 worsens stroke outcomes via cellular fibronectin-EDA but not tenascin C. Obese wild-type mice infused with anti-integrin α9 exhibited improved functional outcomes up to 4 weeks (P<0.05 versus vehicle). CONCLUSIONS: Genetic ablation of neutrophil-specific α9 or pharmacological inhibition improves long-term functional outcomes after stroke in mice with obesity-induced hyperglycemia, most likely by limiting thrombo-inflammation.

Targeting myeloid-cell specific integrin α9ß1 inhibits arterial thrombosis in mice.

Evidence suggests that neutrophils contribute to thrombosis via several mechanisms, including neutrophil extracellular traps (NETs) formation. Integrin α9ß1 is highly expressed on neutrophils when compared with monocytes. It undergoes affinity upregulation on neutrophil activation, and stabilizes adhesion to the activated endothelium. The role of integrin α9 in arterial thrombosis remains unexplored. We generated novel myeloid cell-specific integrin α9-/- mice (α9fl/flLysMCre+) to study the role of integrin α9 in arterial thrombosis. α9fl/fl littermates were used as controls. We report that α9fl/flLysMCre+ mice were less susceptible to arterial thrombosis in ferric chloride (FeCl3) and laser injury-induced thrombosis models with unaltered hemostasis. Neutrophil elastase-positive cells were significantly reduced in α9fl/flLysMCre+ mice concomitant with reduction in neutrophil count, myeloperoxidase levels, and red blood cells in the FeCl3 injury-induced carotid thrombus. The percentage of cells releasing NETs was significantly reduced in α9fl/flLysMCre+ mouse neutrophils stimulated with thrombin-activated platelets. Furthermore, we found a significant decrease in neutrophil-mediated platelet aggregation and cathepsin-G secretion in α9fl/flLysMCre+ mice. Transfusion of α9fl/fl neutrophils in α9fl/flLysMCre+ mice restored thrombosis similar to α9fl/fl mice. Treatment of wild-type mice with anti-integrin α9 antibody inhibited arterial thrombosis. This study identifies the potential role of integrin α9 in modulating arterial thrombosis.

Targeting Myeloid-Specific Integrin α9ß1 Improves Short- and Long-Term Stroke Outcomes in Murine Models With Preexisting Comorbidities by Limiting Thrombosis and Inflammation.

RATIONALE: Currently, there is no effective intervention available that can reduce brain damage following reperfusion. Clinical studies suggest a positive correlation between the increased influx of neutrophils and severity of brain injury following reperfusion. Integrin α9ß1 is highly expressed on activated neutrophils and contributes to stable adhesion, but its role in stroke outcome has not been demonstrated to date. OBJECTIVE: We sought to determine the mechanistic role of myeloid-specific α9ß1 in the progression of ischemic stroke in murine models with preexisting comorbidities. METHODS AND RESULTS: We generated novel myeloid-specific α9-deficient (α9-/-) wild type (α9fl/flLysMCre+/-), hyperlipidemic (α9fl/flLysMCre+/-Apoe-/-), and aged (bone marrow chimeric) mice to evaluate stroke outcome. Susceptibility to ischemia/reperfusion injury was evaluated at 1, 7, and 28 days following reperfusion in 2 models of experimental stroke: filament and embolic. We found that peripheral neutrophils displayed elevated α9 expression following stroke. Irrespective of sex, genetic deletion of α9 in myeloid cells improved short- and long-term stroke outcomes in the wild type, hyperlipidemic, and aged mice. Improved stroke outcome and enhanced survival in myeloid-specific α9-/- mice was because of marked decrease in cerebral thromboinflammatory response as evidenced by reduced fibrin, platelet thrombi, neutrophil, NETosis, and decreased phospho-NF-κB (nuclear factor-κB), TNF (tumor necrosis factor)-α, and IL (interleukin)-1ß levels. α9-/- mice were less susceptible to FeCl3 injury-induced carotid artery thrombosis that was concomitant with improved regional cerebral blood flow following stroke as revealed by laser speckle imaging. Mechanistically, fibronectin containing extra domain A, a ligand for integrin α9, partially contributed to α9-mediated stroke exacerbation. Infusion of a specific anti-integrin α9 inhibitor into hyperlipidemic mice following reperfusion significantly reduced infarct volume and improved short- and long-term functional outcomes up to 28 days. CONCLUSIONS: We provide genetic and pharmacological evidence for the first time that targeting myeloid-specific integrin α9ß1 improves short- and long-term functional outcomes in stroke models with preexisting comorbidities by limiting cerebral thrombosis and inflammation.

Smooth Muscle Cell-Specific PKM2 (Pyruvate Kinase Muscle 2) Promotes Smooth Muscle Cell Phenotypic Switching and Neointimal Hyperplasia.

[Figure: see text].

Differential Roles of Endothelial Cell-Derived and Smooth Muscle Cell-Derived Fibronectin Containing Extra Domain A in Early and Late Atherosclerosis.

OBJECTIVE: The extracellular matrix of atherosclerotic arteries contains abundant deposits of cellular Fn-EDA (fibronectin containing extra domain A), suggesting a functional role in the pathophysiology of atherosclerosis. Fn-EDA is synthesized by several cell types, including endothelial cells (ECs) and smooth muscle cells (SMCs), which are known to contribute to different stages of atherosclerosis. Although previous studies using global Fn-EDA-deficient mice have demonstrated that Fn-EDA is proatherogenic, the cell-specific role of EC versus SMC-derived-Fn-EDA in atherosclerosis has not been investigated yet. Approach and Results: To determine the relative contribution of different pools of Fn-EDA in atherosclerosis, we generated mutant strains lacking Fn-EDA in the ECs (Fn-EDAEC-KO) or smooth muscle cells (Fn-EDASMC-KO) on apolipoprotein E-deficient (Apoe-/-) background. The extent of atherosclerotic lesion progression was evaluated in whole aortae, and cross-sections of the aortic sinus in male and female mice fed a high-fat Western diet for either 4 weeks (early atherosclerosis) or 14 weeks (late atherosclerosis). Irrespective of sex, Fn-EDAEC-KO, but not Fn-EDASMC-KO mice, exhibited significantly reduced early atherogenesis concomitant with decrease in inflammatory cells (neutrophil and macrophage) and VCAM-1 (vascular cell adhesion molecule-1) expression levels within the plaques. In late atherosclerosis model, irrespective of sex, Fn-EDASMC-KO mice exhibited significantly reduced atherogenesis, but not Fn-EDAEC-KO mice, that was concomitant with decreased macrophage content within plaques. Lesional SMCs, collagen content, and plasma inflammatory cytokines (TNF-α [tumor necrosis factor-α] and IL-1ß [interleukin-1ß]), total cholesterol, and triglyceride levels were comparable among groups. CONCLUSIONS: EC-derived Fn-EDA contributes to early atherosclerosis, whereas SMC-derived Fn-EDA contributes to late atherosclerosis.

Fn-EDA (Fibronectin Containing Extra Domain A) in the Plasma, but Not Endothelial Cells, Exacerbates Stroke Outcome by Promoting Thrombo-Inflammation.

Background and Purpose- Cellular Fn-EDA (fibronectin containing extra domain A) is expressed in activated endothelial cells and elevated in circulation in patients with cardiovascular diseases. Although global deficiency of Fn-EDA in mice improves stroke outcome, the specific contribution of plasma versus endothelium Fn-EDA in stroke outcome is currently unknown. We investigated the role of plasma versus endothelial Fn-EDA in stroke exacerbation in the comorbid condition of hyperlipidemia. Methods- We generated novel plasma Fn-EDA-/- ( Fn-EDA fl/fl Alb Cre) and endothelial Fn-EDA-/- ( Fn-EDA fl/fl Tie2 Cre) strains on hyperlipidemic apolipoprotein E-deficient ( ApoE-/-) background. By following the Stroke Therapy Academic Industry Roundtable guidelines, we evaluated stroke outcome in male and female mice. Susceptibility to ischemia/reperfusion injury was evaluated in 2 different models of stroke: intraluminal monofilament and embolic model on days 1, 3, and 7. Quantitative assessment of stroke outcome was evaluated by measuring infarct volume (by magnetic resonance imaging), cerebral blood flow (by laser speckle imaging), neurological and sensory-motor outcome, and postischemic thrombo-inflammation (platelet thrombi, fibrin, neutrophil, phospho-NFκB [nuclear factor κB], TNFα [tumor necrosis factor α], and IL1ß [interleukin 1ß]). Results- Stroke outcome was comparable in ApoE-/- Fn-EDA fl/fl Tie2 Cre and control ApoE-/- Fn-EDA fl/fl mice suggesting endothelial Fn-EDA does not contribute to stroke. ApoE-/- Fn-EDA fl/fl Alb Cre mice exhibited significantly smaller infarcts and improved neurological and sensory-motor outcome at days 1, 3, and 7 in monofilament and embolic models of stroke. Improved stroke outcome was concomitant with enhanced survival, and decreased postischemic thrombo-inflammatory response ( P<0.05 versus ApoE-/- Fn-EDA fl/fl). No sex-based differences were observed. Laser speckle imaging revealed significantly improved regional cerebral blood flow at 1 hour in ApoE-/- Fn-EDA fl/fl Alb Cre mice suggesting plasma Fn-EDA promotes postischemic secondary thrombosis. Coinfusion of anti-Fn-EDA antibody with r-tPA (recombinant tissue-type plasminogen activator) in ApoE-/- mice, 1 hour after embolization, improved stroke outcome with enhanced survival, and improved neurological outcome ( P<0.05 versus r-tPA). Conclusions- Genetic evidence suggests that plasma Fn-EDA exacerbates stroke outcome by promoting postischemic thrombo-inflammation. Interventions targeting plasma Fn-EDA may reduce brain damage after reperfusion.

Fibronectin Containing Extra Domain A Induces Plaque Destabilization in the Innominate Artery of Aged Apolipoprotein E-Deficient Mice.

OBJECTIVE: Fibronectin containing extra domain A (Fn-EDA) is an endogenous ligand of TLR4 (toll-like receptor 4) and is abundant in the extracellular matrix of advanced atherosclerotic lesions in human and mice. Irrespective of sex, deletion of Fn-EDA reduces early atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. However, the contribution of Fn-EDA in advanced atherosclerosis remains poorly characterized. We determined the contribution of Fn-EDA in advanced atherosclerotic lesions of aged (1-year-old) Apoe-/- mice. APPROACH AND RESULTS: Plaque composition was determined in the innominate artery, a plaque instability site that is known to mimic several histological features of vulnerable human plaques. Female Apoe-/-, Fn-EDA-/-Apoe-/-, TLR4-/-Apoe-/-, and Fn-EDA-/-TLR4-/-Apoe-/- mice were fed a high-fat Western diet for 44 weeks. Fn-EDA-/-Apoe-/- mice exhibited reduced plaque size characterized by smaller necrotic cores, thick fibrous caps containing abundant vascular smooth muscle cells and collagen, reduced CD68/MMP9 (matrix metalloproteinase 9)-positive content, less accumulation of MMP-cleaved extracellular matrix aggrecan, and decreased vascular smooth muscle cell and macrophage apoptosis (P<0.05 versus Apoe-/- mice). Together these findings suggest that Fn-EDA induces plaque destabilization. Deletion of TLR4 reduced histological features of plaque instability in Apoe-/- mice but did not further reduce features of plaque destabilization in Fn-EDA-/-Apoe-/- mice, suggesting that TLR4 may contribute to Fn-EDA-induced plaque destabilization. Fn-EDA potentiated TLR4-dependent MMP9 expression in bone marrow-derived macrophages, suggesting that macrophage TLR4 may contribute to Fn-EDA-mediated plaque instability. CONCLUSIONS: Fn-EDA induces histological features of plaque instability in established lesions of aged Apoe-/- mice. The abundance of Fn-EDA in advanced atherosclerotic lesions may increase the risk of plaque destabilization.

Endothelial Cell-Derived Von Willebrand Factor, But Not Platelet-Derived, Promotes Atherosclerosis in Apolipoprotein E-Deficient Mice.

OBJECTIVE: VWF (von Willebrand factor) is synthesized by endothelial cells and megakaryocytes and is known to contribute to atherosclerosis. In vitro studies suggest that platelet-derived VWF (Plt-VWF) is biochemically and functionally different from endothelial cell-derived VWF (EC-VWF). We determined the role of different pools of VWF in the pathophysiology of atherosclerosis. APPROACH AND RESULTS: Using bone marrow transplantation, we generated chimeric Plt-VWF, EC-VWF, and Plt-VWF mice lacking a disintegrin and metalloprotease with thrombospondin type I repeats-13 in platelets and plasma on apolipoprotein E-deficient (Apoe-/-) background. Controls were chimeric Apoe-/- mice transplanted with bone marrow from Apoe-/- mice (wild type) and Vwf-/-Apoe-/- mice transplanted with bone marrow from Vwf-/-Apoe-/- mice (VWF-knock out). Susceptibility to atherosclerosis was evaluated in whole aortae and cross-sections of the aortic sinus in female mice fed a high-fat Western diet for 14 weeks. VWF-knock out, Plt-VWF, and Plt-VWF mice lacking a disintegrin and metalloprotease with thrombospondin type I repeats-13 exhibited reduced plaque size characterized by smaller necrotic cores, reduced neutrophil and monocytes/macrophages content, decreased MMP9 (matrix metalloproteinase), MMP2, and CX3CL1 (chemokine [C-X3-C motif] ligand 1)-positive area, and abundant interstitial collagen (P<0.05 versus wild-type or EC-VWF mice). Atherosclerotic lesion size and composition were comparable between wild-type or EC-VWF mice. Together these findings suggest that EC-VWF, but not Plt-VWF, promotes atherosclerosis exacerbation. Furthermore, intravital microscopy experiments revealed that EC-VWF, but not Plt-VWF, contributes to platelet and leukocyte adhesion under inflammatory conditions at the arterial shear rate. CONCLUSIONS: EC-VWF, but not Plt-VWF, contributes to VWF-dependent atherosclerosis by promoting platelet adhesion and vascular inflammation. Plt-VWF even in the absence of a disintegrin and metalloprotease with thrombospondin type I repeats-13, both in platelet and plasma, was not sufficient to promote atherosclerosis.

ADAMTS13 Retards Progression of Diabetic Nephropathy by Inhibiting Intrarenal Thrombosis in Mice.

OBJECTIVE: ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I repeats-13) prevents microvascular thrombosis by cleaving prothrombogenic ultralarge von Willebrand factor (VWF) multimers. Clinical studies have found association between reduced ADAMTS13-specific activity, ultralarge VWF multimers, and thrombotic angiopathy in patients with diabetic nephropathy. It remains unknown, however, whether ADAMTS13 deficiency or ultralarge VWF multimers have a causative effect in diabetic nephropathy. APPROACH AND RESULTS: The extent of renal injury was evaluated in wild-type (WT), Adamts13-/- and Adamts13-/-Vwf-/- mice after 26 weeks of streptozotocin-induced diabetic nephropathy. We found that WT diabetic mice exhibited low plasma ADAMTS13-specific activity and increased VWF levels (P<0.05 versus WT nondiabetic mice). Adamts13-/- diabetic mice exhibited deterioration of kidney function (increased albuminuria, plasma creatinine, and urea; P<0.05 versus WT diabetic mice), independent of hyperglycemia and hypertension. Deterioration of kidney function in Adamts13-/- diabetic mice was concomitant with aggravated intrarenal thrombosis (assessed by plasminogen activator inhibitor, VWF, fibrin(ogen), and CD41-positive microthrombi), increased mesangial cell expansion, and extracellular matrix deposition (P<0.05 versus WT diabetic mice). Genetic deletion of VWF in Adamts13-/- diabetic mice improved kidney function, inhibited intrarenal thrombosis, and alleviated histological changes in glomeruli, suggesting that exacerbation of diabetic nephropathy in the setting of ADAMTS13 deficiency is VWF dependent. CONCLUSIONS: ADAMTS13 retards progression of diabetic nephropathy, most likely by inhibiting VWF-dependent intrarenal thrombosis. Alteration in ADAMTS13-VWF balance may be one of the key pathophysiological mechanisms of thrombotic angiopathy in diabetes mellitus.

Endothelial Cell-Derived von Willebrand Factor Is the Major Determinant That Mediates von Willebrand Factor-Dependent Acute Ischemic Stroke by Promoting Postischemic Thrombo-Inflammation.

OBJECTIVE: von Willebrand factor (VWF), which is synthesized in endothelial cells and megakaryocytes, is known to worsen stroke outcome. In vitro studies suggest that platelet-derived VWF (Plt-VWF) is biochemically different from the endothelial cell-derived VWF (EC-VWF). However, little is known about relative contribution of different pools of VWF in stroke. APPROACH AND RESULTS: Using bone marrow transplantation, we generated chimeric Plt-VWF mice, Plt-VWF mice that lack ADAMTS13 in platelets and plasma (Plt-VWF/Adamts13(-/-)), and EC-VWF mice to determine relative contribution of different pools of VWF in stroke. In brain ischemia/reperfusion injury model, we found that infarct size and postischemic intracerebral thrombo-inflammation (fibrin(ogen) deposition, neutrophil infiltration, interleukin-1ß, and tumor necrosis factor-α levels) within lesions were comparable between EC-VWF and wild-type mice. Infarct size and postischemic thrombo-inflammation were comparable between Plt-VWF and Plt-VWF/Adamts13(-/-) mice, but decreased compared with EC-VWF and wild-type mice (P<0.05) and increased compared with Vwf(-/-) mice (P<0.05). Susceptibility to FeCl3 injury-induced carotid artery thrombosis was comparable between wild-type and EC-VWF mice, whereas Plt-VWF and Plt-VWF/Adamts13(-/-) mice exhibited defective thrombosis. Although most of the injured vessels did not occlude, slope over time showed that thrombus growth rate was increased in both Plt-VWF and Plt-VWF/Adamts13(-/-) mice compared with Vwf(-/-) mice (P<0.05), but decreased compared with wild-type or EC-VWF mice. CONCLUSIONS: Plt-VWF, either in presence or absence of ADAMTS13, partially contributes to VWF-dependent injury and postischemic thrombo-inflammation after stroke. EC-VWF is the major determinant that mediates VWF-dependent ischemic stroke by promoting postischemic thrombo-inflammation.

Active autophagy but not lipophagy in macrophages with defective lipolysis.

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

Genetic Ablation of Extra Domain A of Fibronectin in Hypercholesterolemic Mice Improves Stroke Outcome by Reducing Thrombo-Inflammation.

BACKGROUND: The fibronectin-splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans but markedly elevated in patients with comorbid conditions, including diabetes mellitus and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe(-/-)) mouse. METHODS AND RESULTS: In a transient cerebral ischemia/reperfusion injury model, Apoe(-/-) mice expressing fibronectin deficient in EDA (Fn-EDA(-/-)Apoe(-/-) mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 versus Apoe(-/-) mice). Concomitantly, intracerebral thrombosis [assessed by fibrin(ogen) deposition] and postischemic inflammation (phospho-nuclear factor-κB p65, phospho-IκB kinase α/ß, interleukin 1ß, and tumor necrosis factor-α) within lesions of Fn-EDA(-/-)Apoe(-/-) mice were markedly decreased (P<0.05 versus Apoe(-/-) mice). In an FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA(-/-)Apoe(-/-) mice (P<0.05 versus Apoe(-/-) mice). Genetic ablation of TLR4 improved stroke outcome in Apoe(-/-) mice (P<0.05) but had no effect on stroke outcome in Fn-EDA(-/-)Apoe(-/-) mice. Bone marrow transplantation experiments revealed that nonhematopoietic cell-derived Fn-EDA exacerbates stroke through Toll-like receptor-4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe(-/-) mouse 15 minutes after reperfusion significantly improved stroke outcome. CONCLUSIONS: Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

Fibronectin Splicing Variants Containing Extra Domain A Promote Atherosclerosis in Mice Through Toll-Like Receptor 4.

OBJECTIVE: Cellular fibronectin containing extra domain A (EDA(+)-FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA(+)-FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA(+)-FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E-deficient (Apoe(-/-)) mouse. APPROACH AND RESULTS: The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA(-/-)Apoe(-/-) mice (which lack EDA(+)-FN), EDA(fl/fl)Apoe(-/-) mice (which constitutively express EDA(+)-FN), and control Apoe(-/-) mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA(fl/fl)Apoe(-/-) mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA(-/-)Apoe(-/-) mice exhibited reduced atherosclerotic lesions (P<0.05 versus Apoe(-/-), n=10-12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA(fl/fl)Apoe(-/-) mice (P<0.05) but had no effect on atherosclerotic lesions in EDA(-/-)Apoe(-/-) mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/NFκB p65, tumor necrosis factor-α, and interleukin-1ß) in bone marrow-derived macrophages from EDA(-/-)Apoe(-/-) mice but not from EDA(-/-)TLR4(-/-)Apoe(-/-) mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA(+)-FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques. CONCLUSIONS: Our findings reveal that TLR4 signaling contributes to EDA(+)-FN-mediated exacerbation of atherosclerosis. We suggest that EDA(+)-FN could be a therapeutic target in atherosclerosis.

Deletion of Methionine Sulfoxide Reductase A Does Not Affect Atherothrombosis but Promotes Neointimal Hyperplasia and Extracellular Signal-Regulated Kinase 1/2 Signaling.

OBJECTIVE: Emerging evidence suggests that methionine oxidation can directly affect protein function and may be linked to cardiovascular disease. The objective of this study was to define the role of the methionine sulfoxide reductase A (MsrA) in models of vascular disease and identify its signaling pathways. APPROACH AND RESULTS: MsrA was readily identified in all layers of the vascular wall in human and murine arteries. Deletion of the MsrA gene did not affect atherosclerotic lesion area in apolipoprotein E-deficient mice and had no significant effect on susceptibility to experimental thrombosis after photochemical injury. In contrast, the neointimal area after vascular injury caused by complete ligation of the common carotid artery was significantly greater in MsrA-deficient than in control mice. In aortic vascular smooth muscle cells lacking MsrA, cell proliferation was significantly increased because of accelerated G1/S transition. In parallel, cyclin D1 protein and cdk4/cyclin D1 complex formation and activity were increased in MsrA-deficient vascular smooth muscle cell, leading to enhanced retinoblastoma protein phosphorylation and transcription of E2F. Finally, MsrA-deficient vascular smooth muscle cell exhibited greater activation of extracellular signal-regulated kinase 1/2 that was caused by increased activity of the Ras/Raf/mitogen-activated protein kinase signaling pathway. CONCLUSIONS: Our findings implicate MsrA as a negative regulator of vascular smooth muscle cell proliferation and neointimal hyperplasia after vascular injury through control of the Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 signaling pathway.

Lack of acyl-CoA:diacylglycerol acyltransferase 1 reduces intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E knockout mice.

Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE(-/-)) mice with Dgat1(-/-) mice. ApoE(-/-) and ApoE(-/-)Dgat1(-/-) mice were fed Western-type diet (WTD) for 9weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE(-/-)Dgat1(-/-) compared with ApoE(-/-) mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE(-/-)Dgat1(-/-) mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.

Intestinal GATA4 deficiency protects from diet-induced hepatic steatosis.

BACKGROUND & AIMS: GATA4, a zinc finger domain transcription factor, is critical for jejunal identity. Mice with an intestine-specific GATA4 deficiency (GATA4iKO) are resistant to diet-induced obesity and insulin resistance. Although they have decreased intestinal lipid absorption, hepatic de novo lipogenesis is inhibited. Here, we investigated dietary lipid-dependent and independent effects on the development of steatosis and fibrosis in GATA4iKO mice. METHODS: GATA4iKO and control mice were fed a Western-type diet (WTD) or a methionine and choline-deficient diet (MCDD) for 20 and 3 weeks, respectively. Functional effects of GATA4iKO on diet-induced liver steatosis were investigated. RESULTS: WTD-but not MCDD-fed GATA4iKO mice showed lower hepatic concentrations of triglycerides, free fatty acids, and thiobarbituric acid reactive species and had reduced expression of lipogenic as well as fibrotic genes compared with controls. Reduced nuclear sterol regulatory element-binding protein-1c protein levels were accompanied by lower lipogenic gene expression. Oil red O and Sirius Red staining of liver sections confirmed the observed reduction in hepatic lipid accumulation and fibrosis. Immunohistochemical staining revealed an increased number of jejunal glucagon-like peptide 1 (GLP-1) positive cells in GATA4iKO mice. Consequently, we found enhanced phosphorylation of hepatic AMP-activated protein kinase and acetyl-CoA carboxylase alpha. CONCLUSIONS: Our results provide strong indications for a protective effect of intestinal GATA4 deficiency on the development of hepatic steatosis and fibrosis via GLP-1, thereby blocking hepatic de novo lipogenesis.

Cellular fibronectin promotes deep vein thrombosis in diet-induced obese mice.

BACKGROUND: Overweight and obesity are significant risk factors for deep vein thrombosis (DVT). Cellular fibronectin containing extra domain A (Fn-EDA), an endogenous ligand for toll-like-receptor 4 (TLR4), contributes to thrombo-inflammation. The role of Fn-EDA in the modulation of DVT is not elucidated yet. OBJECTIVE: To determine whether Fn-EDA promotes DVT in the context of diet-induced obesity. METHODS: Wild-type (WT) and Fn-EDA-deficient mice were either fed control or high-fat (HF) diet for 12 weeks. DVT was induced by inferior vena cava (IVC) stenosis and evaluated after 48 hours. Cellular Fn-EDA levels in the plasma of venous thromboembolism (VTE) patients were measured by sandwich ELISA. RESULTS: We found that cellular Fn-EDA levels were significantly elevated in VTE patients' plasma and positively correlated with body mass index. HF diet-fed WT mice exhibited increased DVT susceptibility compared with control diet-fed WT mice. In contrast, HF diet-fed Fn-EDA-deficient mice exhibited significantly reduced thrombus weight and decreased incidence (%) of DVT compared with HF diet-fed WT mice concomitant with reduced neutrophil content and citrullinated histone H3-positive cells (a marker of NETosis) in IVC thrombus. Exogenous cellular Fn-EDA potentiated NETosis in neutrophils stimulated with thrombin-activated platelets via TLR4. Genetic deletion of TLR4 in Fn-EDA+ mice (constitutively express Fn-EDA in plasma and tissues), but not in Fn-EDA-deficient mice, reduced DVT compared with respective controls. CONCLUSION: These results demonstrate a previously unknown role of Fn-EDA in the DVT exacerbation, which may be an essential mechanism promoting DVT in the setting of diet-induced obesity.

Integrin α9 regulates smooth muscle cell phenotype switching and vascular remodeling.

Excessive proliferation of vascular smooth muscle cells (SMCs) remains a significant cause of in-stent restenosis. Integrins, which are heterodimeric transmembrane receptors, play a crucial role in SMC biology by binding to the extracellular matrix protein with the actin cytoskeleton within the SMC. Integrin α9 plays an important role in cell motility and autoimmune diseases; however, its role in SMC biology and remodeling remains unclear. Herein, we demonstrate that stimulated human coronary SMCs upregulate α9 expression. Targeting α9 in stimulated human coronary SMCs, using anti-integrin α9 antibody, suppresses synthetic phenotype and inhibits SMC proliferation and migration. To provide definitive evidence, we generated an SMC-specific α9-deficient mouse strain. Genetic ablation of α9 in SMCs suppressed synthetic phenotype and reduced proliferation and migration in vitro. Mechanistically, suppressed synthetic phenotype and reduced proliferation were associated with decreased focal adhesion kinase/steroid receptor coactivator signaling and downstream targets, including phosphorylated ERK, p38 MAPK, glycogen synthase kinase 3ß, and nuclear ß-catenin, with reduced transcriptional activation of ß-catenin target genes. Following vascular injury, SMC-specific α9-deficient mice or wild-type mice treated with murine anti-integrin α9 antibody exhibited reduced injury-induced neointimal hyperplasia at day 28 by limiting SMC migration and proliferation. Our findings suggest that integrin α9 regulates SMC biology, suggesting its potential therapeutic application in vascular remodeling.