Glucosylceramide synthase deficiency in the heart compromises β1-adrenergic receptor trafficking

Aims: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function.Methods and results: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors.Conclusions: Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.</p

Energy metabolism and angiogenesis in atherosclerosis and cancer

Energy metabolism requires supply of glucose and oxygen. In atherosclerotic plaques and cancer tumors, there are local areas with mismatch in demand and supply of oxygen and nutrients. The resulting cellular energy imbalance may promote energy failure and stimulation of angiogenesis. In the first paper, energy metabolites were analyzed in human atherosclerotic plaques using high-resolution bioluminescence imaging. Advanced plaques were deficient in ATP and glucose, whereas lactate accumulated. ATP and glucose deficiency was most pronounced in macrophage-rich areas adjacent to the necrotic core. ATP depletion may promote necrotic core expansion and progression from stable to unstable plaques. In the second paper, reactive oxygen species (ROS) production was studied during the development of atherosclerosis in mice. Intracellular ROS levels increased before lesions were visible, suggesting that intracellular ROS promote initiation of atherosclerosis. In advanced atherosclerotic plaques, atorvastatin decreased ROS production in a lipid-lowering independent manner. The decrease in ROS may promote stabilization of plaques. In the third paper, intussusceptive angiogenesis (IA) was demonstrated in human, but not mouse, melanoma metastases. IA may contribute to the growth of human melanoma metastases and help explain the poor effect of current anti-angiogenic drugs targeted to classic sprouting angiogenesis. We further demonstrated that MMP inhibition blocks IA in vitro. In summary, this thesis provides evidence of energy deficiency in human atherosclerotic plaques, new insights into ROS distribution during atherosclerosis development, and finally, evidence of intussusceptive angiogenesis in human malignant melanoma metastases. These data may be used to further the research into better treatments of atherosclerosis and cancer

Human Multilineage 3D Spheroids as a Model of Liver Steatosis and Fibrosis

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries. Despite the high prevalence of NAFLD, the underlying biology of the disease progression is not clear, and there are no approved drugs to treat non-alcoholic steatohepatitis (NASH), the most advanced form of the disease. Thus, there is an urgent need for developing advanced in vitro human cellular systems to study disease mechanisms and drug responses. We attempted to create an organoid system genetically predisposed to NAFLD and to induce steatosis and fibrosis in it by adding free fatty acids. We used multilineage 3D spheroids composed by hepatocytes (HepG2) and hepatic stellate cells (LX-2) with a physiological ratio (24:1). HepG2 and LX-2 cells are homozygotes for the PNPLA3 I148M sequence variant, the strongest genetic determinant of NAFLD. We demonstrate that hepatic stellate cells facilitate the compactness of 3D spheroids. Then, we show that the spheroids develop accumulations of fat and collagen upon exposure to free fatty acids. Finally, this accumulation was rescued by incubating spheroids with liraglutide or elafibranor, drugs that are in clinical trials for the treatment of NASH. In conclusion, we have established a simple, easy to handle, in vitro model of genetically induced NAFLD consisting of multilineage 3D spheroids. This tool may be used to understand molecular mechanisms involved in the early stages of fibrogenesis induced by lipid accumulation. Moreover, it may be used to identify new compounds to treat NASH using high-throughput drug screening

Imaging of Intracellular and Extracellular ROS Levels in Atherosclerotic Mouse Aortas Ex Vivo: Effects of Lipid Lowering by Diet or Atorvastatin

Objective The first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin. To address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-alpha, VCAM, IL-6, and IL-1 beta) or cellular composition (smooth muscle cell, macrophage, and T-cell content). Aortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment

Depletion of ATP and glucose in advanced human atherosclerotic plaques

Objective: Severe hypoxia develops close to the necrotic core of advanced human atherosclerotic plaques, but the energy metabolic consequences of this hypoxia are not known. In animal models, plaque hypoxia is also associated with depletion of glucose and ATP. ATP depletion may impair healing of plaques and promote necrotic core expansion. To investigate if ATP depletion is present in human plaques, we analyzed the distribution of energy metabolites (ATP, glucose, glycogen and lactate) in intermediate and advanced human plaques. Approach and results: Snap frozen carotid endarterectomies from 6 symptomatic patients were analyzed. Each endarterectomy included a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). ATP, glucose, and glycogen concentrations were lower in advanced (ICA) compared to intermediate plaques (CCA), whereas lactate concentrations were higher. The lowest concentrations of ATP, glucose and glycogen were detected in the perinecrotic zone of advanced plaques. Conclusions: Our study demonstrates severe ATP depletion and glucose deficiency in the perinecrotic zone of human advanced atherosclerotic plaques. ATP depletion may impair healing of plaques and promote disease progression

Lack of RAC1 in macrophages protects against atherosclerosis.

The Rho GTPase RAC1 is an important regulator of cytoskeletal dynamics, but the role of macrophage-specific RAC1 has not been explored during atherogenesis. We analyzed RAC1 expression in human carotid atherosclerotic plaques using immunofluorescence and found higher macrophage RAC1 expression in advanced plaques compared with intermediate human atherosclerotic plaques. We then produced mice with Rac1-deficient macrophages by breeding conditional floxed Rac1 mice (Rac1fl/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis was studied in vivo by infecting Rac1fl/fl and Rac1fl/fl/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Rac1fl/fl/LC macrophages secreted lower levels of IL-6 and TNF-α and exhibited reduced foam cell formation and lipid uptake. The deficiency of Rac1 in macrophages reduced the size of aortic atherosclerotic plaques in AdPCSK9-infected Rac1fl/fl/LC mice. Compare with controls, intima/media ratios, the size of necrotic cores, and numbers of CD68-positive macrophages in atherosclerotic plaques were reduced in Rac1-deficient mice. Moreover, we found that RAC1 interacts with actin-binding filamin A. Macrophages expressed increased RAC1 levels in advanced human atherosclerosis. Genetic inactivation of RAC1 impaired macrophage function and reduced atherosclerosis in mice, suggesting that drugs targeting RAC1 may be useful in the treatment of atherosclerosis

Targeting filamin A reduces macrophage activity and atherosclerosis

Background: The actin-binding protein FLNA (filamin A) regulates signal transduction important for cell locomotion, but the role of macrophage-specific FLNA during atherogenesis has not been explored. Methods: We analyzed FLNA expression in human carotid atherosclerotic plaques by immunofluorescence. We also produced mice with Flna-deficient macrophages by breeding conditional Flna-knockout mice (Flna(o/fl)) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis in vivo was studied by transplanting bone marrow from male Flna(o/fl)/LC mice to atherogenic low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice; and by infecting Flna(o/fl) and Flna(o/fl)/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Furthermore, C57BL/6 mice were infected with AdPCSK9 and then treated with the calpain inhibitor calpeptin to inhibit FLNA cleavage. Results: We found that macrophage FLNA expression was higher in advanced than in intermediate human atherosclerotic plaques. Flna(o/fl)/LC macrophages proliferated and migrated less than controls; expressed lower levels of phosphorylated AKT and ERK1/2; exhibited reduced foam cell formation and lipid uptake; and excreted more lipids. The deficiency of Flna in macrophages markedly reduced the size of aortic atherosclerotic plaques in both Ldlr(-/-BMT:Flnao/fl/LC) and AdPCSK9-infected Flna(o/fl)/LC mice. Intima/media ratios and numbers of CD68-positive macrophages in atherosclerotic plaques were lower in Flna-deficient mice than in control mice. Moreover, we found that STAT3 interacts with a calpain-cleaved carboxyl-terminal fragment of FLNA. Inhibiting calpain-mediated FLNA cleavage with calpeptin in macrophages reduced nuclear levels of phosphorylated STAT3, interleukin 6 secretion, foam cell formation, and lipid uptake. Finally, calpeptin treatment reduced the size of atherosclerotic plaques in C57BL/6 mice infected with AdPCSK9. Conclusions: Genetic inactivation of Flna and chemical inhibition of calpain-dependent cleavage of FLNA impaired macrophage signaling and function, and reduced atherosclerosis in mice, suggesting that drugs targeting FLNA may be useful in the treatment of atherosclerosis

Atorvastatin reduces intracellular and extracellular ROS levels within the atherosclerotic aortic arch.

<p>Female <i>Apoe</i>^<i>-/-</i> mice were fed Western diet to induce advanced atherosclerotic lesions in the aortic arch. Then, mice were treated with vehicle (DMSO) or oral atorvastatin (100 mg/kg per day) for 5 days. <i>(A)</i> ROS levels were analyzed in the aortic arch (red areas). <i>(B–D)</i> Atorvastatin treatment reduced intracellular <i>(B and C)</i> and extracellular ROS levels <i>(D)</i>. <i>(E and F)</i> Plasma cholesterol <i>(E)</i> and triglycerides <i>(F)</i> was reduced by atorvastatin and, to larger extent, by lipid lowering by diet. n = 6 in each group. **p<0.01 vs vehicle, *p<0.05 vs vehicle. One sample t-test (C and D). ANOVA with Dunnet’s test for multiple comparisons (E and F).</p

Intracellular ROS correlates with macrophage content and extracellular ROS with smooth muscle cell content in advanced atherosclerotic lesions Female <i>Apoe</i>^<i>-/-</i> mice were fed Western diet to induce advanced atherosclerotic lesions in the aortic arch.

<p>(<i>A)</i> Intracellular and extracellular ROS were analyzed in the aortic arch (red area) <i>(B)</i> Smooth muscle cell and macrophage content was quantified in the aortic arch by analyzing sections from 4 different levels. <i>(C)</i> Section stained for macrophages (CD68: blue) and smooth muscle cells (α-actin: red). <i>(D and E)</i> Smooth muscle cell content in lesions correlated with extracellular <i>(E)</i> but not intracellular ROS <i>(D)</i>. <i>(F and G)</i> Macrophage content in lesions correlated with intracellular <i>(F)</i> but not extracellular ROS <i>(G)</i>. Linear regression (n = 25). NS (non significant).</p