Pyruvate dehydrogenase kinase regulates vascular inflammation in atherosclerosis and increases cardiovascular risk.

AIMS: Recent studies have revealed a close connection between cellular metabolism and the chronic inflammatory process of atherosclerosis. While the link between systemic metabolism and atherosclerosis is well established, the implications of altered metabolism in the artery wall are less understood. Pyruvate dehydrogenase kinase (PDK)-dependent inhibition of pyruvate dehydrogenase (PDH) has been identified as a major metabolic step regulating inflammation. Whether the PDK/PDH axis plays a role in vascular inflammation and atherosclerotic cardiovascular disease remains unclear. METHODS AND RESULTS: Gene profiling of human atherosclerotic plaques revealed a strong correlation between PDK1 and PDK4 transcript levels and the expression of pro-inflammatory and destabilizing genes. Remarkably, the PDK1 and PDK4 expression correlated with a more vulnerable plaque phenotype, and PDK1 expression was found to predict future major adverse cardiovascular events. Using the small-molecule PDK inhibitor dichloroacetate (DCA) that restores arterial PDH activity, we demonstrated that the PDK/PDH axis is a major immunometabolic pathway, regulating immune cell polarization, plaque development, and fibrous cap formation in Apoe-/- mice. Surprisingly, we discovered that DCA regulates succinate release and mitigates its GPR91-dependent signals promoting NLRP3 inflammasome activation and IL-1ß secretion by macrophages in the plaque. CONCLUSIONS: We have demonstrated for the first time that the PDK/PDH axis is associated with vascular inflammation in humans and particularly that the PDK1 isozyme is associated with more severe disease and could predict secondary cardiovascular events. Moreover, we demonstrate that targeting the PDK/PDH axis with DCA skews the immune system, inhibits vascular inflammation and atherogenesis, and promotes plaque stability features in Apoe-/- mice. These results point toward a promising treatment to combat atherosclerosis.

Deep Learning-Based Segmentation of Morphologically Distinct Rat Hippocampal Reactive Astrocytes After Trimethyltin Exposure.

As regulators of homeostasis, astrocytes undergo morphological changes after injury to limit the insult in central nervous system (CNS). Trimethyltin (TMT) is a known neurotoxicant that induces reactive astrogliosis in rat CNS. To evaluate the degree of reactive astrogliosis, the assessment relies on manual counting or semiquantitative scoring. We hypothesized that deep learning algorithm could be used to identify the grade of reactive astrogliosis in immunoperoxidase-stained sections in a quantitative manner. The astrocyte algorithm was created using a commercial supervised deep learning platform and the used training set consisted of 940 astrocytes manually annotated from hippocampus and cortex. Glial fibrillary acidic protein-labeled brain sections of rat TMT model were analyzed for astrocytes with the trained algorithm. Algorithm was able to count the number of individual cells, cell areas, and circumferences. The astrocyte algorithm identified astrocytes with varying sizes from immunostained sections with high confidence. Algorithm analysis data revealed a novel morphometric marker based on cell area and circumference. This marker correlated with the time-dependent progression of the neurotoxic profile of TMT. This study highlights the potential of using novel deep learning-based image analysis tools in neurotoxicity and pharmacology studies.

Genetic Deficiency of Indoleamine 2,3-dioxygenase Aggravates Vascular but Not Liver Disease in a Nonalcoholic Steatohepatitis and Atherosclerosis Comorbidity Model.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that increases cardiovascular disease risk. Indoleamine 2,3-dioxygenase-1 (IDO1)-mediated tryptophan (Trp) metabolism has been proposed to play an immunomodulatory role in several diseases. The potential of IDO1 to be a link between NASH and cardiovascular disease has never been investigated. Using Apoe-/-and Apoe-/-Ido1-/- mice that were fed a high-fat, high-cholesterol diet (HFCD) to simultaneously induce NASH and atherosclerosis, we found that Ido1 deficiency significantly accelerated atherosclerosis after 7 weeks. Surprisingly, Apoe-/-Ido1-/- mice did not present a more aggressive NASH phenotype, including hepatic lipid deposition, release of liver enzymes, and histopathological parameters. As expected, a lower L-kynurenine/Trp (Kyn/Trp) ratio was found in the plasma and arteries of Apoe-/-Ido1-/- mice compared to controls. However, no difference in the hepatic Kyn/Trp ratio was found between the groups. Hepatic transcript analyses revealed that HFCD induced a temporal increase in tryptophan 2,3-dioxygenase (Tdo2) mRNA, indicating an alternative manner to maintain Trp degradation during NASH development in both Apoe-/- and Apoe-/-Ido1-/mice-. Using HepG2 hepatoma cell and THP1 macrophage cultures, we found that iron, TDO2, and Trp degradation may act as important mediators of cross-communication between hepatocytes and macrophages regulating liver inflammation. In conclusion, we show that Ido1 deficiency aggravates atherosclerosis, but not liver disease, in a newly established NASH and atherosclerosis comorbidity model. Our data indicate that the overexpression of TDO2 is an important mechanism that helps in balancing the kynurenine pathway and inflammation in the liver, but not in the artery wall, which likely determined disease outcome in these two target tissues.

Carboxypeptidase A3 expression in canine mast cell tumors and tissue-resident mast cells.

Mast cell tumors (MCTs) are one of the most common cutaneous malignancies in dogs. Previous studies have reported expression of mast cell-specific proteases chymase and tryptase in canine cutaneous MCTs and in connective tissue and mucosal mast cells. In humans and rodents, mast cells express an additional specific protease, carboxypeptidase A3 (CPA3). In this article, we describe CPA3 immunoreactivity in connective tissue, visceral, mucosal, and neoplastic mast cells in dogs. Positive immunolabeling for CPA3 was observed in nonneoplastic mast cells in 20/20 formalin-fixed paraffin-embedded normal tissues (skin, liver, spleen, intestine), and in 63/63 MCTs irrespective of their histological grade. CPA3 protein expression was comparable to that of c-kit in both the nonneoplastic and neoplastic mast cells. Three distinct labeling patterns (membranous, diffuse, and focal cytoplasmic) were observed for CPA3 in MCTs. The focal cytoplasmic labeling pattern was associated with high-grade MCTs staged with the Kiupel 2-tier grading criteria. We propose CPA3 as a novel immunohistochemical marker for canine mast cells in health and disease.

Severe Spontaneous Atherosclerosis in two Korat Breed Cats is Comparable to Human Atherosclerosis.

Atherosclerosis is a chronic inflammatory vascular disease and the leading cause of mortality in humans worldwide. In most domestic animal species, however, primary atherosclerosis is of little clinical relevance. Cats are considered to be atheroresistant and, to our knowledge, spontaneous atherosclerosis has not been reported in cats. Here we report the clinical and histopathological findings in two related cats of the Korat breed that presented with clinical signs of heart failure. In both cases, the clinical signs appeared in adulthood, were progressive and led to death. At necropsy, severe atherosclerotic lesions were present in large and medium-sized arteries and were characterized by the formation of a fibrous cap and a lipid core, which contained a particularly large accumulation of cholesterol crystals, as indicated by the presence of many cholesterol clefts. The lesions closely resembled those of advanced human atherosclerosis. There were no underlying diseases or medical treatments that could have predisposed to the atherosclerosis in these two genetically related cats. A genetic predisposition to human-like atherosclerosis in the local Korat cat population is suspected.

Native and oxidised lipoproteins negatively regulate the serum amyloid A-induced NLRP3 inflammasome activation in human macrophages.

OBJECTIVES: The NLRP3 inflammasome plays a key role in arterial wall inflammation. In this study, we elucidated the role of serum lipoproteins in the regulation of NLRP3 inflammasome activation by serum amyloid A (SAA) and other inflammasome activators. METHODS: The effect of lipoproteins on the NLRP3 inflammasome activation was studied in primary human macrophages and THP-1 macrophages. The effect of oxidised low-density lipoprotein (LDL) was examined in an in vivo mouse model of SAA-induced peritoneal inflammation. RESULTS: Native and oxidised high-density lipoproteins (HDL3) and LDLs inhibited the interaction of SAA with TLR4. HDL3 and LDL inhibited the secretion of interleukin (IL)-1ß and tumor necrosis factor by reducing their transcription. Oxidised forms of these lipoproteins reduced the secretion of mature IL-1ß also by inhibiting the activation of NLRP3 inflammasome induced by SAA, ATP, nigericin and monosodium urate crystals. Specifically, oxidised LDL was found to inhibit the inflammasome complex formation. No cellular uptake of lipoproteins was required, nor intact lipoprotein particles for the inhibitory effect, as the lipid fraction of oxidised LDL was sufficient. The inhibition of NLRP3 inflammasome activation by oxidised LDL was partially dependent on autophagy. Finally, oxidised LDL inhibited the SAA-induced peritoneal inflammation and IL-1ß secretion in vivo. CONCLUSIONS: These findings reveal that both HDL3 and LDL inhibit the proinflammatory activity of SAA and this inhibition is further enhanced by lipoprotein oxidation. Thus, lipoproteins possess major anti-inflammatory functions that hinder the NLRP3 inflammasome-activating signals, particularly those exerted by SAA, which has important implications in the pathogenesis of cardiovascular diseases.

3-Hydroxyanthralinic acid metabolism controls the hepatic SREBP/lipoprotein axis, inhibits inflammasome activation in macrophages, and decreases atherosclerosis in Ldlr-/- mice.

AIMS: Atherosclerosis is a chronic inflammatory disease involving immunological and metabolic processes. Metabolism of tryptophan (Trp) via the kynurenine pathway has shown immunomodulatory properties and the ability to modulate atherosclerosis. We identified 3-hydroxyanthranilic acid (3-HAA) as a key metabolite of Trp modulating vascular inflammation and lipid metabolism. The molecular mechanisms driven by 3-HAA in atherosclerosis have not been completely elucidated. In this study, we investigated whether two major signalling pathways, activation of SREBPs and inflammasome, are associated with the 3-HAA-dependent regulation of lipoprotein synthesis and inflammation in the atherogenesis process. Moreover, we examined whether inhibition of endogenous 3-HAA degradation affects hyperlipidaemia and plaque formation. METHODS AND RESULTS: In vitro, we showed that 3-HAA reduces SREBP-2 expression and nuclear translocation and apolipoprotein B secretion in HepG2 cell cultures, and inhibits inflammasome activation and IL-1ß production by macrophages. Using Ldlr-/- mice, we showed that inhibition of 3-HAA 3,4-dioxygenase (HAAO), which increases the endogenous levels of 3-HAA, decreases plasma lipids and atherosclerosis. Notably, HAAO inhibition led to decreased hepatic SREBP-2 mRNA levels and lipid accumulation, and improved liver pathology scores. CONCLUSIONS: We show that the activity of SREBP-2 and the inflammasome can be regulated by 3-HAA metabolism. Moreover, our study highlights that targeting HAAO is a promising strategy to prevent and treat hypercholesterolaemia and atherosclerosis.

History, clinical findings and outcome of horses with radiographical signs of equine odontoclastic tooth resorption and hypercementosis.

The progression of equine odontoclastic tooth resorption and hypercementosis (EOTRH) has not been completely evaluated, and currently, the only effective treatment is extraction of severely affected teeth. We aim to describe how the disease relates to the history and clinical findings and to report on the outcome in individual horses. This case series comprises data collected from 20 horses (age 14-29 years old) with radiographic findings of EOTRH in their incisor and/or canine teeth. Most horses affected with EOTRH in this study were admitted for dental problems, but some for other complaints such as colic. Of the 288 teeth evaluated radiographically, 224 teeth were abnormal. Radiographic findings were most frequently located in the apical aspect and reserve crown of the teeth, and lesions were also commonly found in clinically normal teeth. Histopathology of extracted teeth showed inflammation in the periodontal ligament and revealed that resorption often extended to the dentine. Some owners were unwilling to allow extraction of their horses' severely affected teeth, even though this treatment has been shown to increase the wellbeing of the horse. As EORTH is a life-long condition, the progression of the disease has to be continuously monitored and the treatments adjusted accordingly.

Chymase released from hypoxia-activated cardiac mast cells cleaves human apoA-I at Tyr192 and compromises its cardioprotective activity.

ApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr192 and Phe229, but hypoxic activation at Tyr192 only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr192-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium.

Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation.

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1ß and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1ß secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1ß secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.

Chronic intermittent psychological stress promotes macrophage reverse cholesterol transport by impairing bile acid absorption in mice.

Psychological stress is a risk factor for atherosclerosis, yet the pathophysiological mechanisms involved remain elusive. The transfer of cholesterol from macrophage foam cells to liver and feces (the macrophage-specific reverse cholesterol transport, m-RCT) is an important antiatherogenic pathway. Because exposure of mice to physical restraint, a model of psychological stress, increases serum levels of corticosterone, and as bile acid homeostasis is disrupted in glucocorticoid-treated animals, we investigated if chronic intermittent restraint stress would modify m-RCT by altering the enterohepatic circulation of bile acids. C57Bl/6J mice exposed to intermittent stress for 5 days exhibited increased transit through the large intestine and enhanced fecal bile acid excretion. Of the transcription factors and transporters that regulate bile acid homeostasis, the mRNA expression levels of the hepatic farnesoid X receptor (FXR), the bile salt export pump (BSEP), and the intestinal fibroblast growth factor 15 (FGF15) were reduced, whereas those of the ileal apical sodium-dependent bile acid transporter (ASBT), responsible for active bile acid absorption, remained unchanged. Neither did the hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), the key enzyme regulating bile acid synthesis, change in the stressed mice. Evaluation of the functionality of the m-RCT pathway revealed increased fecal excretion of bile acids that had been synthesized from macrophage-derived cholesterol. Overall, our study reveals that chronic intermittent stress in mice accelerates m-RCT specifically by increasing fecal excretion of bile acids. This novel mechanism of m-RCT induction could have antiatherogenic potential under conditions of chronic stress.

Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice.

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [(3)H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [(3)H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.