ECP 2023 Abstracts
S126 Virchows Archiv (2023) 483 (Suppl 1):S1–S391 13 Methods: Prospectively sampled five aortic valves of five adult patients referred to the autopsy at the Institute for Pathology, Medical Faculty, University of Belgrade in period of October to Depscember 2022, in whose CAVD was not diagnosed antemortem. The valves were grossly analysed, measured and sampled for microscopic evaluation in order to immunophenotypically assessed osteoid producing cells. Results: There were three male and two female patients with mean age 74.9 (66-88). Grossly measured AV circumference averaged 5.72 cm, (5.4-6 cm), while recalculated AVA (Aortic valve area) averaged 2.62 cm2. On von Kossa special staining were detected multiple diffuse calcified nodules, as well as one larger calcified nodule on all valves. In the vicinity of those calcified nodules and osteogenic matrix were pre- sent spindle cells which immunohistochemical phenotype was in favour of VICs (Vimentin+/CD68-/CD34-), while CD68+ macrophages were distant and peripherally distributed. Endothelial erosion above the cal- cified area, as well as CD34+ areas of neoangiogenesis, were found in 2/5 aortic valves. Conclusion: Nowadays it is known that calcifications in CAVD are not passive and degenerative in nature, but actively produced osteoid matrix. In our study present osteoid producing spindle cells were char- acterized as activated VICs. However, the origin of osteoid producing cells is still under debate and further research in phenotyping of these cells is needed and highly appreciated in order to better understand underlying pathological mechanisms included in CAVD development and possible medical therapeutic approaches. PS-22-003 Cardiovascular mortality and autonomic innervation in myocardial sleeves around pulmonary veins I. Kholova*, D. Depes, A. Mennander, R. Vehniäinen, T. Paavonen *Tampere University and Fimlab Laboratories, Tampere, Finland Background & objectives: Autonomic nerve impairment in the myo- cardium may be associated with an increased risk of cardiovascular morbidity and mortality. We aimed to study autonomic neural remodel- ling in myocardial sleeves around pulmonary veins (PVs) and atrial-PV ostia with immunohistochemistry and clinicopathological correlations. Methods: PVs were collected from 37 and atrial-PV ostia from 17 human autopsy hearts. The density of autonomic nerves was quantified by measuring the area of immunohistochemical staining for sympa- thetic (tyrosine hydroxylase, TH) and parasympathetic (choline acetyl- transferase, CHAT) nerves and ganglia. Growth-associated protein 43 (GAP43) was used as a neural growth marker. Results: In the PV cohort, subjects with immediate cardiovascular cause of death had significantly decreased sympathetic nerve density in fibro-fatty tissue vs. those with non-cardiovascular cause of death (1624.53 μm2/mm2 vs. 2522.05 μm2/mm2, P=0.038). In the atrial-PV ostia cohort, parasympathetic nerve density in myo- cardial sleeves was significantly increased in subjects with underlying cardiovascular cause of death (19.48 μm2/mm2) compared to subjects with underlying non-cardiovascular cause of death (P=0.034). Conclusion: Heterogeneous autonomic innervation around PVs and atrial-PV ostia are associated with cardiovascular morbidity and mortality. Funding: VTR grant, Aarne Koskelo Foundation PS-22-004 Histone methyltransferase DOT1L mediates NLRP3 inflammasome priming and activation in atherosclerotic apolipoprotein E knock- out mice; potential functional implication in human atherosclerosis A. Manea*, M. Vlad, A. Lazar, S. Manea *Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Romania Background & objectives: Alterations in epigenetic mechanisms induce adverse transcriptomic signatures that are associated with the pathoetiology of atherosclerotic disease. The study aimed at elucidat- ing the potential implication of DOT1L epigenetic enzyme in medi- ating NLRP3 inflammasome priming and activation in experimental atherosclerosis. Methods: Non-atherosclerotic and atherosclerotic tissue specimens derived from patients undergoing extended carotid endarterectomy, ApoE-/- mice and polarized pro-inflammatory (M1)/anti-inflamma- tory/(M2) human macrophages (Mac) were examined employing immunofluorescence microscopy, real-time PCR and Western blot. ApoE-/- mice fed a normal/atherogenic diet were treated with 5 mg/kg EPZ004777, a specific DOT1L inhibitor, or vehicle, for 4 weeks. Rest- ing and M1/M2-Mac underwent EPZ pharmacological interventions. Results: The mRNA and protein levels of DOT1L, NLRP3, caspase 1 (Cas1), IL1β and IL18 were found significantly elevated in human atherosclerotic lesions, atherosclerotic aorta of ApoE-/- mice and in cultured M1-Mac. The colocalization of DOT1L and NLRP3 inflam- masome components with lesional Mac within both human and mouse atherosclerotic plaques was demonstrated by immunofluorescence microscopy. Systemic DOT1L blockade led to significant decreases in mRNA and protein levels of NLRP3, Cas1, IL1β, and IL18 in the atherosclerotic aorta of ApoE-/- mice. Inhibition of DOT1L suppressed the mRNA and protein up-regulation of NLRP3, Cas1, IL1β and IL18 in cultured pro-inflammatory M1-Mac. Conclusion: DOT1L-induced histone H3 methylation (H3K79me1/me2/ me3) generates long-lasting transcriptomic alterations, since unlike other histone methyltransferases, no specific demethylases of these histone modifications have been characterized. Activation of NLRP3 inflam- masome leading to increased production of IL1β and IL18 is instrumen- tal in atherogenesis. We provide evidence that DOT1L contributes to NLRP3 inflammasome priming and activation in both in vivo and in vitro experimental settings of atherosclerosis. The data point to DOT1L as a promising therapeutic target to reduce inflammation in atherosclerosis. This work was supported in part by grants from the Ministry of Research, Innovation and Digitization, CNCS – UEFISCDI (PN-III- P4-ID-PCE-2020-1898, PN-III-P1-1.1-TE-2021-0180), within PNCDI III and from the Romanian Academy. PS-22-005 Pharmacological inhibition of histone methyltransferase SET7 reduces inflammation and fibrosis in the kidney of diabetic mice S. Manea*, A. Lazar, M. Vlad, A. Manea *Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Romania Background & objectives: Inflammation and fibrosis are important mechanisms leading to renal dysfunction and ultimately kidney failure in diabetic kidney disease (DKD). We aimed to uncover the potential role of histone methyltransferase SET7 in mediating pro-inflammatory and pro-fibrotic responses in diabetic kidney. Methods: Male non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were treated with 5 mg/kg (R)-PFI 2 hydrochloride, a specific SET7 inhibitor, or its vehicle for 4 weeks. Human endothelial cells (EA.hy926, EC) underwent PFI pharmacological interventions and transient transfection assays employing a human SET7 expression vector. Histochemistry, immunofluorescence microscopy, real-time PCR, and Western blot techniques were used to examine the samples. Results: Significantly elevated mRNA/protein levels of SET7, pro- inflammatory cell adhesion molecules (E-selectin, ICAM-1, VCAM- 1), extracellular matrix proteins (collagen IV/Col IV, fibronectin/FN, laminin/LM), and pro-fibrotic factor TGFβ were determined in the kid- ney of diabetic mice. SET7 blockade significantly reduced glomerular hypertrophy, and the gene and protein expression levels of E-selectin, ICAM-1, VCAM-1, Col IV, FN, LM and TGFβ in diabetic kidney. The expression of SET7 was significantly induced in high glucose- exposed EC. PFI intervention suppressed the high glucose-induced
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