Enzymatic defluorination of a terminally monofluorinated pentyl moiety: oxidative or hydrolytic mechanism?

Fluorination of organic compounds plays an important role in the chemical and pharmaceutical industry and is often applied in order to improve physicochemical parameters or modify pharmacological properties. While oxidative and reductive defluorination have been shown to be responsible for the metabolic degradation of organofluorine compounds, the involvement of hydrolytic mechanisms catalyzed by human enzymes has not been reported so far. Here, we investigated the enzymatic defluorination of terminally monofluorinated aliphates with [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM-2201) as a model substance. We performed in vitro biotransformation using pooled human liver microsomes (pHLM) and human recombinant cytochrome P450 (CYP) assays. In order to elucidate the underlying mechanisms, modified incubation conditions were applied including the use of deuterium labeled AM-2201 (d2 -AM-2201). Identification of the main metabolites and analysis of their isotopic composition was performed by liquid-chromatography coupled to time-of-flight-mass-spectrometry (LC-QToF-MS). Quantification of the metabolites was achieved with a validated method based on liquid-chromatography-tandem-mass-spectrometry (LC-MS/MS). CYP 1A2 mediated defluorination of d2 -AM-2201 revealed an isotopic pattern of the defluorinated 5-hydroxypentyl metabolite (5-HPM) indicating a redox mechanism with an aldehyde as a plausible intermediate. In contrast, formation of 5-HPM by pHLM was observed independently of the presence of atmospheric oxygen or co-factors regenerating the redox system. pHLM incubation of d2 -AM-2201 confirmed the hypothesis of a non-oxidative mechanism involved in the defluorination of the 5-fluoropentyl moiety. So far, enzymatically catalyzed, hydrolytic defluorination was only described in bacteria and other prokaryotes. The presented data prove the involvement of a hydrolytic mechanism catalyzed by human microsomal enzymes other than CYP. Significance Statement Elucidating the mechanisms involved in the enzymatic detoxification of organofluorine compounds is crucial for enhancing our understanding and facilitating the design and development of drugs with improved pharmacokinetic profiles. The carbon-fluorine bond possesses a high binding energy, which suggests that non-activated fluoroalkanes would not undergo hydrolytic cleavage. However, our study provides evidence for the involvement of a non-oxidative mechanism catalyzed by human liver enzymes. It is important to consider CYP-independent, hydrolytic defluorination, when investigating the pharmacokinetic properties of fluorinated xenobiotics.

Genetic Deficiency of TRAF5 Promotes Adipose Tissue Inflammation and Aggravates Diet-Induced Obesity in Mice.

Objective: The accumulation of inflammatory leukocytes is a prerequisite of adipose tissue inflammation during cardiometabolic disease. We previously reported that a genetic deficiency of the intracellular signaling adaptor TRAF5 (TNF [tumor necrosis factor] receptor-associated factor 5) accelerates atherosclerosis in mice by increasing inflammatory cell recruitment. Here, we tested the hypothesis that an impairment of TRAF5 signaling modulates adipose tissue inflammation and its metabolic complications in a model of diet-induced obesity in mice. Approach and Results: To induce diet-induced obesity and adipose tissue inflammation, wild-type or Traf5-/- mice consumed a high-fat diet for 18 weeks. Traf5-/- mice showed an increased weight gain, impaired insulin tolerance, and increased fasting blood glucose. Weight of livers and peripheral fat pads was increased in Traf5-/- mice, whereas lean tissue weight and growth were not affected. Flow cytometry of the stromal vascular fraction of visceral adipose tissue from Traf5-/- mice revealed an increase in cytotoxic T cells, CD11c+ macrophages, and increased gene expression of proinflammatory cytokines and chemokines. At the level of cell types, expression of TNF[alpha], MIP (macrophage inflammatory protein)-1[alpha], MCP (monocyte chemoattractant protein)-1, and RANTES (regulated on activation, normal T-cell expressed and secreted) was significantly upregulated in Traf5-deficient adipocytes but not in Traf5-deficient leukocytes from visceral adipose tissue. Finally, Traf5 expression was lower in adipocytes from obese patients and mice and recovered in adipose tissue of obese patients one year after bariatric surgery. Conclusions: We show that a genetic deficiency of TRAF5 in mice aggravates diet-induced obesity and its metabolic derangements by a proinflammatory response in adipocytes. Our data indicate that TRAF5 may promote anti-inflammatory and obesity-preventing signaling events in adipose tissue.

Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice.

OBJECTIVES: The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaques of apolipoprotein E-deficient (Apoe -/- ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCreERT2-driven) deficiency of CD40 in Apoe -/- mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. CONCLUSION: Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering.

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression.

Residual inflammatory risk in coronary heart disease: incidence of elevated high-sensitive CRP in a real-world cohort.

BACKGROUND: Inflammation drives atherosclerosis and its complications. Anti-inflammatory therapy with interleukin 1 beta (IL-1ß) antibody reduces cardiovascular events in patients with elevated high-sensitive C-reactive protein (hsCRP). This study aims to identify the share of patients with coronary heart disease (CHD) and residual inflammation who may benefit from anti-inflammatory therapy. METHODS: hsCRP and low-density lipoprotein (LDL) levels were determined in 2741 all-comers admitted to the cardiological ward of our tertiary referral hospital between June 2016 and June 2018. Patients without CHD, with acute coronary syndrome, chronic or recurrent systemic infection, use of immunosuppressant or anti-inflammatory agents, chronic inflammatory diseases, chemotherapy, terminal organ failure, traumatic injury and pregnancy were excluded. RESULTS: 856 patients with stable CHD were included. 42.7% of those had elevated hsCRP ≥ 2 mg/l. Within the group of patients with LDL-cholesterol < 70 mg/dl, 30.9% shared increased hsCRP indicating residual inflammation. After multivariate adjusted backward selection elevated Lipoprotein (a) (OR 1.61, p = 0.048), elevated proBNP (OR 2.57, p < 0.0001), smoking (OR 1.70, p = 0.022), and obesity (OR 2.28, p = 0.007) were associated with elevated hsCRP. In contrast, the use of ezetimibe was associated with normal hsCRP (OR 0.51, p = 0.014). In the subgroup of patients with on-target LDL-cholesterol < 70 mg/dl, backward selection identified elevated proBNP (OR 3.49, p = 0.007) as independent predictor of elevated hsCRP in patients with LDL-cholesterol < 70 mg/dl. CONCLUSION: One-third of all-comers patients with CHD showed increased levels of hsCRP despite a LDL-cholesterol < 70 mg/dl potentially qualifying for an anti-inflammatory therapy. Elevated proBNP is an independent risk factor for hsCRP elevation.

Glucose lowering by SGLT2-inhibitor empagliflozin accelerates atherosclerosis regression in hyperglycemic STZ-diabetic mice.

Diabetes worsens atherosclerosis progression and leads to a defect in repair of arteries after cholesterol reduction, a process termed regression. Empagliflozin reduces blood glucose levels via inhibition of the sodium glucose cotransporter 2 (SGLT-2) in the kidney and has been shown to lead to a marked reduction in cardiovascular events in humans. To determine whether glucose lowering by empagliflozin accelerates atherosclerosis regression in a mouse model, male C57BL/6J mice were treated intraperitoneally with LDLR- and SRB1- antisense oligonucleotides and fed a high cholesterol diet for 16 weeks to induce severe hypercholesterolemia and atherosclerosis progression. At week 14 all mice were rendered diabetic by streptozotocin (STZ) injections. At week 16 a baseline group was sacrificed and displayed substantial atherosclerosis of the aortic root. In the remaining mice, plasma cholesterol was lowered by switching to chow diet and treatment with LDLR sense oligonucleotides to induce atherosclerosis regression. These mice then received either empagliflozin or vehicle for three weeks. Atherosclerotic plaques in the empagliflozin treated mice were significantly smaller, showed decreased lipid and CD68+ macrophage content, as well as greater collagen content. Proliferation of plaque resident macrophages and leukocyte adhesion to the vascular wall were significantly decreased in empagliflozin-treated mice. In summary, plasma glucose lowering by empagliflozin improves plaque regression in diabetic mice.

Tumor Necrosis Factor Receptor-Associated Factor 5 Promotes Arterial Neointima Formation through Smooth Muscle Cell Proliferation.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are cytoplasmic adaptor proteins of the TNF/interleukin (IL)-1/Toll-like receptor superfamily. Ligands of this family such as TNFα, CD40L, and IL-1ß promote chronic inflammatory processes such as atherosclerosis and restenosis, the latter being a common adverse reaction after vascular interventions. We previously reported overexpression of TRAF5 in murine and human atheromata and TRAF5-dependent proinflammatory functions in vitro. However, the role of TRAF5 in restenosis remains unsettled. To evaluate whether TRAF5 affects neointima formation, TRAF5-/-LDLR-/- and TRAF5+/+LDLR-/- mice consuming a high cholesterol diet (HCD) received wire-induced injury of the carotid artery. After 28 days, TRAF5-deficient mice showed a 45% decrease in neointimal area formation compared with TRAF5-compentent mice. Furthermore, neointimal vascular smooth muscle cells (vSMC) and macrophages decreased whereas collagen increased in TRAF5-deficient mice. Mechanistically, the latter expressed lower transcript levels of the matrix metalloproteinases 2 and 9, both instrumental in extracellular matrix degradation and vSMC mobilization. Additionally, TRAF5-specific siRNA interference rendered murine vSMC less proliferative upon CD40L stimulation. In accordance with these findings, fewer vSMC isolated from TRAF5-deficient aortas were in a proliferative state as assessed by Ki67 and cyclin B1 expression. In conclusion, TRAF5 deficiency mitigates neointima formation in mice, likely through a TRAF5-dependent decrease in vSMC proliferation.

Purinergic receptor Y2 (P2Y2)- dependent VCAM-1 expression promotes immune cell infiltration in metabolic syndrome.

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y2. The gene expression of ATP receptor P2Y2 did not change in several tissues in the course of obesity, but was increased within epididymal fat. Adipose tissue from P2Y 2-/- mice consuming high-fat diet (HFD) contained less crown-like structures with a reduced frequency of adipose tissue macrophages (ATMs). This was likely due to decreased leukocyte migration because of missing VCAM-1 exposition on P2Y2 deficient hypertrophic adipose tissue endothelial cells. Accordingly, P2Y 2-/- mice showed blunted traits of the metabolic syndrome: they gained less weight compared to P2Y 2+/+ controls, while intake of food and movement behaviour remained unchanged. Liver and adipose tissue were smaller in P2Y 2-/- animals. Insulin tolerance testing (ITT) performed in obese P2Y 2-/- mice revealed a better insulin sensitivity as well as lower plasma C-peptide and cholesterol levels. We demonstrate that interfering with somatic P2Y2 signalling prevents excessive immune cell deposition in diet-induced obesity (DIO), both attenuating adipose tissue inflammation and ameliorating the metabolic phenotype. Thus, blocking the P2Y2 cascade may be a promising strategy to limit metabolic disease and its sequelae.

Endothelial cell CD36 optimizes tissue fatty acid uptake.

Movement of circulating fatty acids (FAs) to parenchymal cells requires their transfer across the endothelial cell (EC) barrier. The multiligand receptor cluster of differentiation 36 (CD36) facilitates tissue FA uptake and is expressed in ECs and parenchymal cells such as myocytes and adipocytes. Whether tissue uptake of FAs is dependent on EC or parenchymal cell CD36, or both, is unknown. Using a cell-specific deletion approach, we show that EC, but not parenchymal cell, CD36 deletion increased fasting plasma FAs and postprandial triglycerides. EC-Cd36-KO mice had reduced uptake of radiolabeled long-chain FAs into heart, skeletal muscle, and brown adipose tissue; these uptake studies were replicated using [11C]palmitate PET scans. High-fat diet-fed EC-CD36-deficient mice had improved glucose tolerance and insulin sensitivity. Both EC and cardiomyocyte (CM) deletion of CD36 reduced heart lipid droplet accumulation after fasting, but CM deletion did not affect heart glucose or FA uptake. Expression in the heart of several genes modulating glucose metabolism and insulin action increased with EC-CD36 deletion but decreased with CM deletion. In conclusion, EC CD36 acts as a gatekeeper for parenchymal cell FA uptake, with important downstream effects on glucose utilization and insulin action.

A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense.

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.

Novel Reversible Model of Atherosclerosis and Regression Using Oligonucleotide Regulation of the LDL Receptor.

RATIONALE: Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and methods that block liver lipoprotein secretion. OBJECTIVE: We sought to develop a more direct or time-effective method to create and then reverse hypercholesterolemia and atherosclerosis via transient knockdown of the hepatic LDLR (low-density lipoprotein receptor) followed by its rapid restoration. METHODS AND RESULTS: We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild-type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels because of recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, that is, regression of inflammatory cell content, after a period of 2 to 3 weeks. CONCLUSIONS: We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. Although cholesterol reduction decreased early en face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.

Inflammatory Pathways Regulated by Tumor Necrosis Receptor-Associated Factor 1 Protect From Metabolic Consequences in Diet-Induced Obesity.

RATIONALE: The coincidence of inflammation and metabolic derangements in obese adipose tissue has sparked the concept of met-inflammation. Previous observations, however, suggest that inflammatory pathways may not ultimately cause dysmetabolism. OBJECTIVE: We have revisited the relationship between inflammation and metabolism by testing the role of TRAF (tumor necrosis receptor-associated factor)-1, an inhibitory adapter of inflammatory signaling of TNF (tumor necrosis factor)-α, IL (interleukin)-1ß, and TLRs (toll-like receptors). METHODS AND RESULTS: Mice deficient for TRAF-1, which is expressed in obese adipocytes and adipose tissue lymphocytes, caused an expected hyperinflammatory phenotype in adipose tissue with enhanced adipokine and chemokine expression, increased leukocyte accumulation, and potentiated proinflammatory signaling in macrophages and adipocytes in a mouse model of diet-induced obesity. Unexpectedly, TRAF-1-/- mice were protected from metabolic derangements and adipocyte growth, failed to gain weight, and showed improved insulin resistance-an effect caused by increased lipid breakdown in adipocytes and UCP (uncoupling protein)-1-enabled thermogenesis. TRAF-1-dependent catabolic and proinflammatory cues were synergistically driven by ß3-adrenergic and inflammatory signaling and required the presence of both TRAF-1-deficient adipocytes and macrophages. In human obesity, TRAF-1-dependent genes were upregulated. CONCLUSIONS: Enhancing TRAF-1-dependent inflammatory pathways in a gain-of-function approach protected from metabolic derangements in diet-induced obesity. These findings identify TRAF-1 as a regulator of dysmetabolism in mice and humans and question the pathogenic role of chronic inflammation in metabolism.

P2X7 Deficiency Blocks Lesional Inflammasome Activity and Ameliorates Atherosclerosis in Mice.

BACKGROUND: Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2X7 and promotes inflammasome assembly and interleukin-1ß expression. We hypothesized a functional role of the signal axis ATP-P2X7 in inflammasome activation and the chronic inflammation driving atherosclerosis. METHODS: P2X7-competent and P2X7-deficient macrophages were isolated and stimulated with lipopolysaccharide, ATP, or both. To assess whether P2X7 may have a role in atherosclerosis, P2X7 expression was analyzed in aortic arches from low density lipoprotein receptor-/- mice consuming a high-cholesterol or chow diet. P2X7+/+ and P2X7-/- low density lipoprotein receptor-/- mice were fed a high-cholesterol diet to investigate the functional role of P2X7 knockout in atherosclerosis. Human plaques were derived from carotid endarterectomy and stained against P2X7. RESULTS: Lipopolysaccharide or ATP stimulation alone did not activate caspase 1 in isolated macrophages. However, priming with lipopolysaccharide, followed by stimulation with ATP, led to an activation of caspase 1 and interleukin-1ß in P2X7-competent macrophages. In contrast, P2X7-deficient macrophages showed no activation of caspase 1 after sequential stimulation while still expressing a basal amount of interleukin-1ß. P2X7 receptor was higher expressed in murine atherosclerotic lesions, particularly by lesional macrophages. After 16 weeks of a high-cholesterol diet, P2X7-deficient mice showed smaller atherosclerotic lesions than P2X7-competent mice (0.162 cm2±0.023 [n=9], P2X7-/- low density lipoprotein receptor-/- : 0.084 cm2±0.01 [n=11], P=0.004) with a reduced amount of lesional macrophages. In accord with our in vitro findings, lesional caspase 1 activity was abolished in P2X7-/- mice. In addition, intravital microscopy revealed reduced leukocyte rolling and adhesion in P2X7-deficient mice. Last, we observe increased P2X7 expression in human atherosclerotic lesions, suggesting that our findings in mice are relevant for human disease. CONCLUSIONS: P2X7 deficiency resolved plaque inflammation by inhibition of lesional inflammasome activation and reduced experimental atherosclerosis. Therefore, P2X7 represents an interesting potential new target to combat atherosclerosis.

Extracellular ATP Induces Vascular Inflammation and Atherosclerosis via Purinergic Receptor Y2 in Mice.

OBJECTIVE: A solid body of evidence supports a role of extracellular ATP and its P2 receptors in innate and adaptive immunity. It promotes inflammation as a danger signal in various chronic inflammatory diseases. Thus, we hypothesize contribution of extracellular ATP and its receptor P2Y2 in vascular inflammation and atherosclerosis. APPROACH AND RESULTS: Extracellular ATP induced leukocyte rolling, adhesion, and migration in vivo as assessed by intravital microscopy and in sterile peritonitis. To test the role of extracellular ATP in atherosclerosis, ATP or saline as control was injected intraperitoneally 3× a week in low-density lipoprotein receptor(-/-) mice consuming high cholesterol diet. Atherosclerosis significantly increased after 16 weeks in ATP-treated mice (n=13; control group, 0.26 mm2; ATP group, 0.33 mm2; P=0.01). To gain into the role of ATP-receptor P2Y2 in ATP-induced leukocyte recruitment, ATP was administered systemically in P2Y2-deficient or P2Y2-competent mice. In P2Y2-deficient mice, the ATP-induced leukocyte adhesion was significantly reduced as assessed by intravital microscopy. P2Y2 expression in atherosclerosis was measured by real-time polymerase chain reaction and immunohistochemistry and demonstrates an increased expression mainly caused by influx of P2Y2-expressing macrophages. To investigate the functional role of P2Y2 in atherogenesis, P2Y2-deficient low-density lipoprotein receptor(-/-) mice consumed high cholesterol diet. After 16 weeks, P2Y2-deficient mice showed significantly reduced atherosclerotic lesions with decreased macrophages compared with P2Y2-competent mice (n=11; aortic arch: control group, 0.25 mm(2); P2Y2-deficient, 0.14 mm2; P=0.04). Mechanistically, atherosclerotic lesions from P2Y2-deficient mice expressed less vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 RNA. CONCLUSIONS: We show that extracellular ATP induces vascular inflammation and atherosclerosis via activation of P2Y2.

Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression.

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6C(high) monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe(-/-) mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6C(high) monocytes and macrophages. SYK inhibition limited Ly6C(high) monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe(-/-) mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.

Cardiac Myocyte KLF5 Regulates Ppara Expression and Cardiac Function.

RATIONALE: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. OBJECTIVE: To determine the role of Krüppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. METHODS AND RESULTS: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte-specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism-related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. CONCLUSIONS: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.

Effects of High Fat Feeding and Diabetes on Regression of Atherosclerosis Induced by Low-Density Lipoprotein Receptor Gene Therapy in LDL Receptor-Deficient Mice.

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr-/- mice in different metabolic states.

Lipolysis, and not hepatic lipogenesis, is the primary modulator of triglyceride levels in streptozotocin-induced diabetic mice.

OBJECTIVE: Diabetic hypertriglyceridemia is thought to be primarily driven by increased hepatic de novo lipogenesis. However, experiments in animal models indicated that insulin deficiency should decrease hepatic de novo lipogenesis and reduce plasma triglyceride levels. APPROACH AND RESULTS: To address the discrepancy between human data and genetically altered mouse models, we investigated whether insulin-deficient diabetic mice had triglyceride changes that resemble those in diabetic humans. Streptozotocin-induced insulin deficiency increased plasma triglyceride levels in mice. Contrary to the mouse models with impaired hepatic insulin receptor signaling, insulin deficiency did not reduce hepatic triglyceride secretion and de novo lipogenesis-related gene expression. Diabetic mice had a marked decrease in postprandial triglycerides clearance, which was associated with decreased lipoprotein lipase and peroxisome proliferator-activated receptor α mRNA levels in peripheral tissues and decreased lipoprotein lipase activity in skeletal muscle, heart, and brown adipose tissue. Diabetic heterozygous lipoprotein lipase knockout mice had markedly elevated fasting plasma triglyceride levels and prolonged postprandial triglycerides clearance. CONCLUSIONS: Insulin deficiency causes hypertriglyceridemia by decreasing peripheral lipolysis and not by an increase in hepatic triglycerides production and secretion.

Coinhibitory suppression of T cell activation by CD40 protects against obesity and adipose tissue inflammation in mice.

BACKGROUND: Costimulatory cascades such as the CD40L-CD40 dyad enhance immune cell activation and inflammation during atherosclerosis. Here, we tested the hypothesis that CD40 directly modulates traits of the metabolic syndrome in diet-induced obesity in mice. METHODS AND RESULTS: To induce the metabolic syndrome, wild-type or CD40(-/-) mice consumed a high-fat diet for 20 weeks. Unexpectedly, CD40(-/-) mice exhibited increased weight gain, impaired insulin secretion, augmented accumulation of inflammatory cells in adipose tissue, and enhanced proinflammatory gene expression. This proinflammatory and adverse metabolic phenotype could be transplanted into wild-type mice by reconstitution with CD40-deficient lymphocytes, indicating a major role for CD40 in T or B cells in this context. Conversely, therapeutic activation of CD40 signaling by the stimulating antibody FGK45 abolished further weight gain during the study, lowered glucose levels, improved insulin sensitivity, and suppressed adipose tissue inflammation. Mechanistically, CD40 activation decreased the expression of proinflammatory cytokines in T cells but not in B cells or macrophages. Finally, repopulation of lymphocyte-free Rag1(-/-) mice with CD40(-/-) T cells provoked dysmetabolism and inflammation, corroborating a protective role of CD40 on T cells in the metabolic syndrome. Finally, levels of soluble CD40 showed a positive association with obesity in humans, suggesting clinical relevance of our findings. CONCLUSIONS: We present the surprising finding that CD40 deficiency on T cells aggravates whereas activation of CD40 signaling improves adipose tissue inflammation and its metabolic complications. Therefore, positive modulation of the CD40 pathway might describe a novel therapeutic concept against cardiometabolic disease.

Successful therapy of ventricular rupture by percutaneous intrapericardial instillation of fibrin glue: a case report.

Rupture of the ventricular myocardium is an often lethal complication after myocardial infarction. Due to the dramatic hemodynamics and the short time frame between ventricular rupture and surgical closure of the defect, additional therapeutic strategies are needed. Here we report the successful therapy of ventricular rupture by percutaneous intrapericardial instillation of fibrin glue in a 72-year-old male patient with postinfarct angina secondary to anterior myocardial infarction.