Association between C10X polymorphism in the CARD8 gene and inflammatory markers in young healthy individuals in the LBA study.

BACKGROUND: The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity as a negative regulator of NF- ĸB, and has been associated with regulation of proteins involved in inflammation. Expression of CARD8 mRNA and protein has been identified in human atherosclerotic lesions, and the truncated T30A variant (rs2043211) of CARD8 has been associated with lower C-reactive (CRP) and MCP-1 levels in myocardial infarction patients. The present study examines the role of a genetic variation in the CARD8 gene in relation to a selection of markers of inflammation. METHODS: In a cross-sectional study of young healthy individuals (18.0-25.9 yrs, n = 744) the association between the rs2043211 variant in the CARD8 gene and protein markers of inflammation was assessed. Genotyping of the CARD8 C10X (rs2043211) polymorphism was performed with TaqMan real time PCR on DNA from blood samples. Protein levels were studied via Olink inflammation panel ( https://olink.com/ ). Using linear models, we analyzed men and two groups of women with and without estrogen containing contraceptives separately, due to previous findings indicating differences between estrogen users and non-estrogen using women. Genotypes were analyzed by additive, recessive and dominant models. RESULTS: The minor (A) allele of the rs2043211 polymorphism in the CARD8 gene was associated with lower levels of CCL20 and IL-6 in men (CCL20, Additive model: p = 0.023; Dominant model: p = 0.016. IL-6, Additive model: p = 0.042; Dominant model: p = 0.039). The associations remained significant also after adjustment for age and potential intermediate variables. CONCLUSIONS: Our data indicate that CARD8 may be involved in the regulation of CCL20 and IL-6 in men. No such association was observed in women. These findings strengthen and support previous in vitro data on IL-6 and CCL20 and highlight the importance of CARD8 as a factor in the regulation of inflammatory proteins. The reason to the difference between sexes is however not clear, and the influence of estrogen as a possible factor important for the inflammatory response needs to be further explored.

Targeting serotonin receptor 2B inhibits TGFß induced differentiation of human vascular smooth muscle cells.

Vascular Smooth Muscle Cells (VSMCs) are known to be the key drivers of intimal thickening which contribute to early progression of atherosclerosis. VSMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli they could modify the type of matrix proteins produced. Serotonin receptor 2B (5-HT2B receptor/HTR2B) has been implicated in several chronic fibrotic and vascular diseases. Although studies have successfully demonstrated the efficacy of HTR2B blockade in attenuating fibrotic disease, the role of 5-HT2B receptor in TGFß mediated VSMC differentiation remain largely unknown. In the present study, we investigated the potential of targeting the 5-HT2B receptor to prevent TGFß induced VSMCs differentiation. Our results showed that 5-HT2B receptors are expressed in human atherosclerotic lesion and HTR2B expression positively correlated to the VSMCs markers. We show that AM1125, a selective 5-HT2B receptor inhibitor, significantly inhibits TGFß1 induced production of collagen and CTGF. The investigation of underlying mechanisms indicated that 5-HT2B receptor antagonism blocks phospho-Smad2 mediated downstream signaling of TGFß1 in vascular smooth muscle cells. Collectively, the HTR2B/TGF-ß1/Phospho-Smad2 pathway plays a critical role in the regulation of VSMCs differentiation. Our findings might serve 5-HT2B receptor as a therapeutic target to limit TGF-ß1 induced VSMC differentiation.

Novel purine analogues regulate IL-1ß release via inhibition of JAK activity in human aortic smooth muscle cells.

Purine analogues bearing a nitrate ester motif were previously discovered as cardioprotective and anti-inflammatory agents, but the anti-inflammatory mechanism remains to be established. We therefore investigated the anti-inflammatory effect of two purine analogues, MK118 bearing a nitrate ester moiety and the methyl-substituted analogue MK196 in Aortic Smooth Muscle Cells (AoSMCs), with emphasis on IL-1ß release. The AoSMCs were stimulated with LPS with or without purine analogue, followed by ELISA, Olink proteomics, Western blot and real time PCR of NLRP3 inflammasome components. Both purine analogues inhibited the release of proteins involved in inflammation, such as TRAIL, CCL4, CSF1 and IL-1ß in AoSMCs, as well as intracellular gene and protein expression of IL-1ß and NLRP3 inflammasome components. MK196, but not MK118, also inhibited the LPS-induced release of IL-7, CXCL10, PD-L1, FLT3L and CCL20. We also showed that MK118 and possibly MK196 act via inhibition of JAKs. In silico studies showed that the purine moiety is a competent hinge binding motif and that the purine-piperazine scaffold is well accommodated in the lipophilic groove of JAK1-3. Both compounds establish interactions with catalytic amino acids in the active site of JAK1-3 and the terminal nitrate ester of MK118 was revealed as a promising pharmacophore. Our data suggest that MK118 and MK196 inhibit the release of proinflammatory proteins in AoSMCs, and targets JAK1-3 activation. Purine analogues also inhibit the expression of NLRP3 inflammasome genes and proteins and may in the future be evaluated for anti-inflammatory aspects on inflammatory diseases.

Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells.

The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity and overexpression of CARD8 mRNA was previously identified in atherosclerosis. However, very little is known about the regulation of CARD8 in endothelial cells and atherosclerosis. The aim of this study was to investigate CARD8 in the regulation of cytokine and chemokine expression in endothelial cells. Sections of human atherosclerotic lesions and non-atherosclerotic arteries were immunostained for CARD8 protein. Expression of CARD8 was correlated to mediators of inflammation in atherosclerotic lesions using Biobank of Karolinska Endarterectomies microarray data. The CARD8 mRNA was knocked-down in human umbilical vein endothelial cells (HUVECs) in vitro, followed by quantitative RT-PCR analysis and OLINK Proteomics. Endothelial and smooth muscle cells in arterial tissue expressed CARD8 and CARD8 correlated with vWF, CD163 and the expression of inflammatory genes, such as CXCL1, CXCL6 and PDGF-A in plaque. Knock-down of CARD8 in HUVECs significantly altered proteins involved in inflammatory response, such as CXCL1, CXCL6, PDGF-A, MCP-1 and IL-6. The present study suggest that CARD8 regulate the expression of cytokines and chemokines in endothelial cells and atherosclerotic lesions, suggesting that CARD8 plays a significant role in endothelial activation.

A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition.

Natural purines like ATP, ADP and adenosine have crucial roles in platelet physiology. This knowledge has been significant in drug development and today ADP receptor antagonists are widely used for prevention of thrombotic events following myocardial infarction and ischaemic stroke. Recent studies have shown that a purine analogue bearing nitrate ester group (denoted MK128) has anti-inflammatory effects probably due to its ability to donate nitric oxide (NO). However, other pharmacological mechanisms may contribute to the observed effect. The aim of the present study was to establish the anti-platelet activity and elucidate the underlying molecular mechanism(s) of the purine analogue MK128. We found that MK128 reduced aggregation and secretion induced by the thrombin receptor agonist SFLLRN and nearly abolished aggregation and secretion induced by thromboxane A2 (TxA2) and collagen receptor agonists. The inhibition took place despite blockage of the NO/cGMP signalling system. Furthermore, interaction between MK128 and platelet purinergic receptors did not explain the observed inhibition. Instead, we found that MK128 concentration-dependently inhibited Rho-associated kinase (ROCK), which led to decreased ROCK-dependent myosin phosphatase target subunit (MYPT)-1 phosphorylation and suppression of platelet functional responses.

Lysophosphatidic Acid Signaling in Obesity and Insulin Resistance.

Although simple in structure, lysophosphatidic acid (LPA) is a potent bioactive lipid that profoundly influences cellular signaling and function upon binding to G protein-coupled receptors (LPA1-6). The majority of circulating LPA is produced by the secreted enzyme autotaxin (ATX). Alterations in LPA signaling, in conjunction with changes in autotaxin (ATX) expression and activity, have been implicated in metabolic and inflammatory disorders including obesity, insulin resistance, and cardiovascular disease. This review summarizes our current understanding of the sources and metabolism of LPA with focus on the influence of diet on circulating LPA. Furthermore, we explore how the ATX-LPA pathway impacts obesity and obesity-associated disorders, including impaired glucose homeostasis, insulin resistance, and cardiovascular disease.