To Örebro University

Proinflammatory cytokine interleukin (IL)-1β is a key mediator of the inflammatory response in atherosclerosis. Targeting IL-1β represents a new approach for the anti-inflammatory therapy of cardiovascular diseases. Based on our previous data demonstrating that cardioprotective 6-piperazinyl purines can effectively inhibit IL-1β release in vascular cells, in this study, we present the synthesis of a next generation of purine analogues bearing either a furoxan moiety as a nitric oxide (NO) donor, or a (methylsulfonyl)thio group, a benzothioamide, or a 5-phenyl-3H-1,2-dithiole-3-thione as putative hydrogen sulfide (H2S) donor moieties. NO and H2S are gaseous signaling molecules that can reduce vascular inflammation. The new purine analogues were evaluated for their anti-inflammatory activity by assessing their inhibitory effect on the secretion of lipopolysaccharide (LPS)-induced IL-1β in human aortic smooth muscle cells (HAoSMCs). Our initial findings revealed that compounds bearing a [methylsulfonyl)thio]propanoyl or [methylsulfonyl)thio]hexanoyl group (compounds MK175 and MK169, respectively) could effectively inhibit LPS-induced IL-1β release in HAoSMCs.

BACKGROUND AND PURPOSE: Pharmacological intervention of thrombosis is challenging, requiring a fined tune balance between beneficial antithrombotic effect versus risk of major bleeding complications. In this investigation, we elucidated the antithrombotic capacity of the novel 90-mer RNA aptamer Apta-1 and its underlying mechanism of action.

EXPERIMENTAL APPROACH: We utilized three independent in vivo animal models to establish antithrombotic activity and bleeding risk of Apta-1. Several cellular and molecular techniques were utilized to extensively characterize the effects of Apta-1 on primary and secondary haemostasis.

KEY RESULTS: Apta-1 significantly reduced thrombus weight in ferric chloride-induced carotid artery thrombosis. A consistent reduction in thrombus weight was also observed in arteriovenous shunt thrombosis in rats, whereas tail bleeding time was unaffected. Cellular and molecular analyses revealed that Apta-1 interacted with thrombin, resulting in significant inhibition of protease-activated receptor (PAR) signalling in platelets. On the other hand, Apta-1 shortened both thrombin generation and thrombin-induced clotting times.

CONCLUSIONS AND IMPLICATIONS: Apta-1 targets the heparin-binding motif exosite II on thrombin leading to significant suppression of platelet PAR1 and PAR4 signalling. Intriguingly, Apta-1 produces substantial antithrombotic activity without anticoagulant or general antiplatelet properties. In fact, we found that Apta-1 accelerates the formation of blood clots and thus supports haemostasis without exhibiting typical anticoagulant properties. We suggest that Apta-1 may be a promising future drug candidate for treatment of thrombosis in diseases/conditions where there are significant risks of serious bleeding complications.

BACKGROUND: Blockade of IL1RAP (interleukin 1 receptor associated protein) was recently shown to reduce atherosclerosis in mice, but the effect on human vascular cells is largely unknown. Targeting the IL1RAP coreceptor represents a novel strategy to block the IL1RAP-dependent cytokines IL (interleukin)-1, IL-33, and IL-36. In the present study, we aimed to evaluate the role of novel antibodies targeting IL1RAP to reduce the effects of IL-1β, IL-33, or IL-36γ in human vascular cells.

METHODS: Expression of IL1RAP was observed in human atherosclerotic plaques by immunohistochemistry and microarray and in endothelial cells by flow cytometry. Endothelial cells were cultured with IL-1β, IL-33, or IL-36γ cytokines with or without IL1RAP antibodies and analyzed with Olink proteomics, ELISA, Western blot, and real-time quantitative polymerase chain reaction. The functional effect of IL1RAP antibodies on endothelial cells were analyzed with adhesion and permeability assays.

RESULTS: Olink proteomics showed inhibition of the inflammatory proteins LIF (leukemia inhibitory factor), OPG (osteoprotegerin), CCL4 (C-C motif chemokine ligand 4), and MCP-3 (monocyte chemoattractant protein 3) by IL1RAP-blockade in endothelial cells after IL-1β stimulation. In addition, the IL1RAP antibodies inhibited IL-1β, and IL-33 induced IL-6 and IL-8 secretion. Secretion of MCP-1 (monocyte chemoattractant protein 1) was induced by IL-1β, IL-33, and IL-36γ, and subsequently was inhibited by IL1RAP antibodies. Similar effects were found on mRNA expression level. Endothelial expression of the adhesion markers ICAM1, VCAM1, and SELE were significantly reduced by IL1RAP antibodies, and neutrophil adhesion to endothelial cells induced by IL-1β and IL-33 was reduced by IL1RAP blockade. In human atherosclerotic lesions, IL1RAP expression correlated with markers of inflammation like IL6, IL8, and MCP1.

CONCLUSIONS: IL1RAP-targeting antibodies can reduce the expression of inflammatory cytokines and markers of adhesion in endothelial cells, which may be of importance for future putative targeted treatments against cardiovascular disease.

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.

Introduction: Nitrate ester bearing 6-piperazinyl-purine analogues (denoted MK drugs) are cardioprotective in infarction animal models and act as inhibitors of Janus kinase (JAK) and Rho-associated kinase (ROCK) [1-3]. Despite the presence of nitrate ester moiety, the MK drugs do not release nitric oxide (NO).

Methods: We utilized organic chemistry platforms to design a dinitrate ester derivative denoted MK177, cell-free and cellular assays to elucidate antithrom-botic and anti-ischemic mechanisms. Furthermore, we also used tissue models to analyze vasodilation, and animal models to evaluate drug activities in vivo.

Results: In anesthetized pigs, intravenous infusion of MK177 produced“nitroglycerin-like”effects on vital parameters. Analysis of exhaled air confirmed release of NO. MK177 caused concentration-dependent relaxation of iliac arteries (87±6.8 % relaxation of precontracted arteries, mean value ±SD, n=5) and this effect was mediated by activation of the NO/cyclic GMP signaling pathway. It is noteworthy that other mononitrate or non-nitrate MKs did not cause NO-induced vasodilation. In cellular model systems, MK177 evoked antithrombotic effects by targeting ROCK in a NO-independent manner. Specifically, MK177 inhibited platelet aggregation induced by collagen (72±12.6 % inhibition of aggregation, mean value±SD, n=7). Western blot analyses confirmed that MK177 reduced ROCK-dependent phosphorylation of myosin phosphatase sub-unit (MYPT-1) in platelets. Finally, kinase screening assay revealed that MK177 concentration-dependently inhibited ROCK and JAK (Kd values around 5μM).

Conclusion: We have developed a bifunctional drug molecule, MK177, that acts by NO-dependent and NO-independent mechanisms. MK 177 induced car-diovascular NO effects in vivo and relaxed vessels in vitro. MK177 also prevented blood platelet activation via NO-independent ROCK inhibition. The bifunctional nature of MK177 can be of significance in future management of thrombotic and ischemic disease. Collectively, the novel cardio-protective and bifunctional drug MK177 has promising therapeutic potential.

References:

1. Koufaki M, Fotopoulou T, Iliodromitis EK, Bibli SI, Zoga A, Kremastinos DT, Andreadou I. Discovery of 6-[4-(6-nitroxyhexanoyl)(piperazin-1-yl)]-9H-purine, as pharmacological post-conditioning agent. Bioorg Med Chem. 2012;20(19):5948-5956. https://doi.org/10.1016/j.bmc.2012.07.037

2. Kardeby C, Paramel GV, Pournara D, Fotopoulou T, Sirsjö A, Koufaki M, Fransén K, Grenegård M. A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition. Eur J Pharmacol. 2019;15(857):172428-172434. https://doi.org/10.1016/j.ejphar.2019.172428

3. Paramel GV, Lindkvist M, Idosa BA, Sebina LS, Kardeby C, Fotopoulou T, Pournara D, Kritsi E, Ifanti E, Zervou M, Koufaki M, Grenegård M, Fransén K. Novel purine analogues regulate IL-1βrelease via inhibition of JAK activity in human aortic smooth muscle cells. Eur J Pharmacol. 2022;15(929):175128-175135. https://doi.org/10.1016/j.ejphar.2022.175128