1.8-cineole prevents platelet activation and aggregation by activating the cAMP pathway via the adenosine A2A receptor.

AIMS: Dysregulated platelet aggregation is a fatal condition in many bacterial- and virus-induced diseases. However, classical antithrombotics cannot completely prevent immunothrombosis, due to the unaddressed mechanisms towards inflammation. Thus, targeting platelet hyperactivation together with inflammation might provide new treatment options in diseases, characterized by immunothrombosis, such as COVID-19 and sepsis. The aim of this study was to investigate the antiaggregatory effect and mode of action of 1.8-cineole, a monoterpene derived from the essential oil of eucalyptus leaves, known for its anti-inflammatory proprieties. MAIN METHODS: Platelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA. KEY FINDINGS: 1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole. SIGNIFICANCE: Given the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.

Isothiocyanates in medicine: A comprehensive review on phenylethyl-, allyl-, and benzyl-isothiocyanates.

In recent years, isothiocyanates (ITCs), bioactive compounds primarily derived from Brassicaceae vegetables and herbs, have gained significant attention within the biomedical field due to their versatile biological effects. This comprehensive review provides an in-depth exploration of the therapeutic potential and individual biological mechanisms of the three specific ITCs phenylethyl isothiocyanate (PEITC), allyl isothiocyanate (AITC), and benzyl isothiocyanate (BITC), as well as their collective impact within the formulation of ANGOCIN® Anti-Infekt N (Angocin). Angocin comprises horseradish root (Armoracia rusticanae radix, 80 mg) and nasturtium (Tropaeoli majoris herba, 200 mg) and is authorized for treating inflammatory diseases affecting the respiratory and urinary tract. The antimicrobial efficacy of this substance has been confirmed both in vitro and in various clinical trials, with its primary effectiveness attributed to ITCs. PEITC, AITC, and BITC exhibit a wide array of health benefits, including potent anti-inflammatory, antioxidant, and antimicrobial properties, along with noteworthy anticancer potentials. Moreover, we highlight their ability to modulate critical biochemical pathways, such as the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and signal transducer and activator of transcription (STAT) pathways, shedding light on their involvement in cellular apoptosis and their intricate role to guide immune responses.

Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion.

AIMS: Arrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool. METHODS AND RESULTS: IgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), Western blot analysis and Triton X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs. CONCLUSION: Our study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC.

Cardiomyocyte cohesion is increased after ADAM17 inhibition.

A Disintegrin And Metalloprotease (ADAM) family proteins are involved in several cardiac diseases, and some ADAMs have been associated with cardiomyopathies. ADAM17 is known to cleave desmoglein 2 (DSG2), one of the proteins involved in the pathogenesis of arrhythmogenic cardiomyopathy (AC). Desmosomal stability is impaired in AC, an inheritable genetic disease, the underlying causes of which can be mutations in genes coding for proteins of the desmosome, such as DSG2, desmoplakin (DP), plakoglobin (PG), plakophilin 2 or desmocollin 2. Stabilizing desmosomal contacts can therefore be a treatment option. In the heart of the murine Jup -/- AC model, (Jup being the gene coding for PG) mice, elevated levels of p38MAPK, an activator of ADAM17, were found. However, ADAM17 levels were unaltered in Jup -/- mice hearts. Nonetheless, inhibition of ADAM17 led to enhanced cardiomyocyte cohesion in both Jup +/+ and Jup -/- mice, and in HL-1 cardiomyocytes. Further, enhanced cohesion in HL-1 cardiomyocytes after acute inhibition of ADAM17 was paralleled by enhanced localization of DSG2 and DP at the membrane, whereas no changes in desmosomal assembly or the desmosomal complex were observed. In conclusion, acute inhibition of ADAM17 might lead to reduced cleavage of DSG2, thereby stabilizing the desmosomal adhesion, evidenced by increased DSG2 and DP localization at cell borders and eventually cardiomyocyte cohesion. We believe that similar mechanisms exist in AC.

EGFR inhibition leads to enhanced desmosome assembly and cardiomyocyte cohesion via ROCK activation.

Arrhythmogenic cardiomyopathy (AC) is a familial heart disease partly caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity may provide new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of the epidermal growth factor receptor (EGFR) in cardiomyocyte cohesion. We inhibited EGFR under physiological and pathophysiological conditions using the murine plakoglobin-KO AC model, in which EGFR was upregulated. EGFR inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and atomic force microscopy (AFM) revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. PamGene Kinase assay performed in HL-1 cardiomyocytes treated with erlotinib, an EGFR inhibitor, revealed upregulation of Rho-associated protein kinase (ROCK). Erlotinib-mediated desmosome assembly and cardiomyocyte cohesion were abolished upon ROCK inhibition. Thus, inhibiting EGFR and, thereby, stabilizing desmosome integrity via ROCK might provide treatment options for AC.

Cardiomyocyte adhesion and hyperadhesion differentially require ERK1/2 and plakoglobin.

Arrhythmogenic cardiomyopathy (AC) is a heart disease often caused by mutations in genes coding for desmosomal proteins, including desmoglein-2 (DSG2), plakoglobin (PG), and desmoplakin (DP). Therapy is based on symptoms and limiting arrhythmia, because the mechanisms by which desmosomal components control cardiomyocyte function are largely unknown. A new paradigm could be to stabilize desmosomal cardiomyocyte adhesion and hyperadhesion, which renders desmosomal adhesion independent from Ca2+. Here, we further characterized the mechanisms behind enhanced cardiomyocyte adhesion and hyperadhesion. Dissociation assays performed in HL-1 cells and murine ventricular cardiac slice cultures allowed us to define a set of signaling pathways regulating cardiomyocyte adhesion under basal and hyperadhesive conditions. Adrenergic signaling, activation of PKC, and inhibition of p38MAPK enhanced cardiomyocyte adhesion, referred to as positive adhesiotropy, and induced hyperadhesion. Activation of ERK1/2 paralleled positive adhesiotropy, whereas adrenergic signaling induced PG phosphorylation at S665 under both basal and hyperadhesive conditions. Adrenergic signaling and p38MAPK inhibition recruited DSG2 to cell junctions. In PG-deficient mice with an AC phenotype, only PKC activation and p38MAPK inhibition enhanced cardiomyocyte adhesion. Our results demonstrate that cardiomyocyte adhesion can be stabilized by different signaling mechanisms, which are in part offset in PG-deficient AC.