T Lymphocyte-Derived Exosomes Transport MEK1/2 and ERK1/2 and Induce NOX4-Dependent Oxidative Stress in Cardiac Microvascular Endothelial Cells

Background: Activation of endothelial cells by inflammatory mediators secreted by CD4+ T lymphocytes plays a key role in the inflammatory response. Exosomes represent a specific class of signaling cues transporting a mixture of proteins, nucleic acids, and other biomolecules. So far, the impact of exosomes shed by T lymphocytes on cardiac endothelial cells remained unknown. Methods and results: Supernatants of CD4+ T cells activated with anti-CD3/CD28 beads were used to isolate exosomes by differential centrifugation. Activation of CD4+ T cells enhanced exosome production, and these exosomes (CD4-exosomes) induced oxidative stress in cardiac microvascular endothelial cells (cMVECs) without affecting their adhesive properties. Furthermore, CD4-exosome treatment aggravated the generation of mitochondrial reactive oxygen species (ROS), reduced nitric oxide (NO) levels, and enhanced the proliferation of cMVECs. These effects were reversed by adding the antioxidant apocynin. On the molecular level, CD4-exosomes increased NOX2, NOX4, ERK1/2, and MEK1/2 in cMVECs, and ERK1/2 and MEK1/2 proteins were found in CD4-exosomes. Inhibition of either MEK/ERK with U0126 or ERK with FR180204 successfully protected cMVECs from increased ROS levels and reduced NO bioavailability. Treatment with NOX1/4 inhibitor GKT136901 effectively blocked excessive ROS and superoxide production, reversed impaired NO levels, and reversed enhanced cMVEC proliferation triggered by CD4-exosomes. The siRNA-mediated silencing of Nox4 in cMVECs confirmed the key role of NOX4 in CD4-exosome-induced oxidative stress. To address the properties of exosomes under inflammatory conditions, we used the mouse model of CD4+ T cell-dependent experimental autoimmune myocarditis. In contrast to exosomes obtained from control hearts, exosomes obtained from inflamed hearts upregulated NOX2, NOX4, ERK1/2, MEK1/2, increased ROS and superoxide levels, and reduced NO bioavailability in treated cMVECs, and these changes were reversed by apocynin. Conclusion: Our results point to exosomes as a novel class of bioactive factors secreted by CD4+ T cells in immune response and represent potential important triggers of NOX4-dependent endothelial dysfunction. Neutralization of the prooxidative aspect of CD4-exosomes could open perspectives for the development of new therapeutic strategies in inflammatory cardiovascular diseases

Influence of Particle Size and Extraction Methods on Phenolic Content and Biological Activities of Pear Pomace

The main goal of this research was to investigate how particle size influences the characteristics of pear (Pyrus Communis L.) pomace flour and to examine the impact of different pre-treatment methods on the phenolic content and associated bioactivities. Pear pomace flour was fractionated into different particle sizes, namely 1 mm, 710 m, 180 m, 75 m and 53 m. Then two extraction methods, namely maceration with methanol and two-step extraction with hexane via Soxhlet followed by ultrasound extraction with methanol, were tested. Total phenolic and total flavonoid contents ranged from 375.0 to 512.9 mg gallic acid/100 g DW and from 24.7 to 34.6 mg quercetin/100 g DW, respectively. Two-step extraction provided antioxidant activity up to 418.8 (in FRAP assay) and 340.0 mg Trolox/100 g DW (in DPPH assay). In order to explore various bioactive properties, this study assessed the inhibitory effects of enzymes, specifically -amylase and -glucosidase (associated with antidiabetic effects), as well as angiotensin-converting enzyme (linked to potential antihypertensive benefits). Additionally, the research investigated antibacterial potential against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, revealing significant results (p < 0.05), particularly in the case of the two-step extraction method. This investigation underscores the substantial value of certain food industry wastes, highlighting their potential as bioactive ingredients within the framework of a circular economy.info:eu-repo/semantics/publishedVersio

Brachypodium distachyon - a model plant to study grass genome structure, dynamics and evolution

Plenary Session: Present status of cell, tissue and organ in vitro cultur

Anti-Oxidant and Anti-Enzymatic Activities of Sea Buckthorn (Hippophaë rhamnoides L.) Fruits Modulated by Chemical Components

The aim of this study was to analyze in vitro biological activities as anti-oxidant, anti-&alpha;-amylase, anti-&alpha;-glucosidase, anti-lipase, and anti-lipoxygenase activity, relative to bioactive components (phenolic acids, flavonols, xanthophylls, carotenes, esterified carotenoids, tocopherols, tocotrienols, and fatty acids) and the basic chemical composition (sugars, organic acid, dry matter, soluble solid, pH, titratable acidity, ash, pectins, and vitamin C) of Hippopha&euml; rhamnoides berries. Six sea buckthorn cultivars commonly grown in Poland were analyzed including Aromatnaja, Botaniczeskaja-Lubitelskaja, J&oacute;zef, Luczistaja, Moskwiczka, and Podarok Sadu. Berries contained 1.34&ndash;2.87 g of sugars and 0.96&ndash;4.22 g of organic acids in 100 g fresh weight, 468.60&ndash;901.11 mg of phenolic compounds, and 46.61&ndash;508.57 mg of carotenoids in 100 g dry mass. The fatty acid profile was established: palmitic &gt; palmitoleic &gt; oleic and linoleic &gt; stearic and linolenic acids. The highest anti-oxidant (34.68 mmol Trolox/100 g dry mass) and anti-&alpha;-amylase potential (IC50 = 26.83 mg/mL) was determined in Aromatnaja, anti-&alpha;-glucosidase in Botaniczeskaja-Lubitelskaja (IC50 = 41.78 mg/mL), anti-lipase in Moskwiczka and Aromatnaja (average IC50 = 4.37 mg/mL), and anti-lipoxygenase in Aromatnaja and Podarok Sadu fruits (100% inhibition). The studied sea buckthorn berries may be a raw material for the development of functional foods and nutraceutical products rich in compounds with high biological activity

Expression analysis of selected TGF-β signaling pathway components in children with transient hypogammaglobulinemia of infancy

Niedobory odporności przebiegające z przewagą zaburzeń produkcji przeciwciał stanowią największą grupę pierwotnych niedoborów odporności (ang. primary immunodeficiencies disease, PID). Jednym z nich jest przejściowa hipogammaglobulinemia wieku dziecięcego (ang. transient hypogammaglobulinemia of infancy, THI), która charakteryzuje się obniżonym poziomem IgG (czasami również IgA i/lub IgM) oraz nawracającymi infekcjami. Wstępne rozpoznanie THI stwierdzane jest po wykluczeniu innych znanych przyczyn hipogammaglobulinemii, przy czym definitywna diagnoza ustalana jest wyłącznie retrospektywnie, po normalizacji poziomu immunoglobulin w surowicy oraz stanu klinicznego chorych, co następuje zazwyczaj pomiędzy 2 a 5 rokiem życia. Patomechanizm leżący u podłoża tego niedoboru pozostaje nieznany. Biorąc pod uwagę najnowsze doniesienia wskazujące na istotną rolę podwyższonej liczby limfocytów T regulatorowych (ang. regulatory T cells, Treg) w rozwoju THI, celem niniejszej pracy była próba określenia ewentualnych zaburzeń w obrębie kanonicznego szlaku przekazu sygnału od TGF-β promującego ekspresję czynnika Foxp3 – swoistego markera tych komórek. Grupę badaną utworzyli pacjenci z THI, jak również zdrowe osoby, u których metodą cytofluorymetryczną oceniano poziom krążących limfocytów Treg. W kolejnym etapie, z pozyskanych od pacjentów komórek jednojądrzastych krwi obwodowej (ang. peripheral blood mononuclear cells, PBMC) izolowano RNA. Poziomy ekspresji poszczególnych genów kodujących składowe kanonicznego szlaku sygnałowania związanego z czynnikiem TGF-β, oceniano metodą PCR w czasie rzeczywistym z użyciem barwnika SYBR Green.W wyniku przeprowadzonych badań, nie wykazano związku pomiędzy poziomem ekspresji genów dla obu izoform czynnika TGF-β jak i jego receptorów a liczebnością krążących limfocytów Treg, co może sugerować istnienie innego mechanizmu warunkującego podwyższoną liczbę tych komórek w przebiegu THI. Z kolei, obniżona ekspresja genu SMAD3, obserwowana w grupie dzieci z wyrównaną formą THI, może sugerować możliwy mechanizm normalizacji poziomu limfocytów Treg u dzieci z tym niedoborem odporności.Predominantly antibody deficiencies are the most common forms of primary immunodeficiency diseases (PID). One of them, transient hypogammaglobulinemia of infancy (THI) is a heterogeneous disorder characterized by reduced serum IgG (and sometimes IgA and/or IgM) level in the early childhood and recurrent infections. A putative diagnosis is initially made after exclusion of other causes of hypogammaglobulinemia, while a definitive diagnosis of THI can only be made retrospectively in patients with normalized IgG levels and withdrawal of clinical symptoms that occurs usually between 2nd and 5th year of life. The underlying definitive basis of this disorder is still unknown. Based on the latest reports concerning the possible role of elevated number of T regulatory cells (Treg) in THI patients, the aim of this study was the evaluation of possible defects in canonical TGF-β signaling pathway that promote expression of transcription factor Foxp3 – a specific marker of these cells.Children with THI, as well as healthy subjects, were enrolled in whom the absolute number of circulating Treg cells was determined using flow cytometry. Total RNA was isolated from peripheral blood mononuclear cells. To determined the expression profile of selected pathway component of TGF-β signaling cascade, real-time SYBR Green PCR assay was used.As a result, no relationship between both isoforms of TGF-β genes expression levels, as well as its receptors, and the number of circulating Treg cells was observed. It may suggest that there is another mechanism that determines the elevated number of these cells in THI patients. Furthermore, reduced expression of the SMAD3 gene observed in children with definitive diagnosis of THI suggest a possible mechanism of Treg cells normalization in this patients

Crosstalk between the TGF-β and WNT signalling pathways during cardiac fibrogenesis

Cardiac fibrosis is referred to as an excessive accumulation of stromal cells and extracellular matrix proteins in the myocardium. Progressive fibrosis causes stiffening of the cardiac tissue and affects conduction of electrical impulses, leading to heart failures in a broad range of cardiac conditions. At the cellular level, activation of the cardiac stromal cells and myofibroblast formation are considered as hallmarks of fibrogenesis. At the molecular level, transforming growth factor β (TGF-β) is traditionally considered as a master regulator of the profibrotic processes. More recently, the WNT signalling pathway has also been found to be implicated in the development of myocardial fibrosis. In this review, we summarize current knowledge on the involvement of TGF-β and WNT downstream molecular pathways to cardiac fibrogenesis and describe a crosstalk between these two profibrotic pathways. TGF-β and WNT ligands bind to different receptors and trigger various outputs. However, a growing body of evidence points to cross-regulation between these two pathways. It has been recognized that in cardiac pathologies TGF-β activates WNT/β-catenin signalling, which in turn stabilizes the TGF-β/Smad response. Furthermore both, the non-canonical TGF-β and non-canonical WNT signalling pathways, activate the same mitogen-activated protein kinases (MAPKs): the extracellular signal-regulated kinase (Erk), the c-Jun N-terminal kinases (JNKs) and p38. The crosstalk between TGF-β and WNT pathways seems to play an essential role in switching on the genetic machinery initiating profibrotic changes in the heart. Better understanding of these mechanisms will open new opportunities for development of targeted therapeutic approaches against cardiac fibrosis in the future

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases

The Types of Polysaccharide Coatings and Their Mixtures as a Factor Affecting the Stability of Bioactive Compounds and Health-Promoting Properties Expressed as the Ability to Inhibit the α-Amylase and α-Glucosidase of Chokeberry Extracts in the Microencapsulation Process

This study aimed to evaluate the feasibility of microencapsulating chokeberry extract by extrusion, and assess the effects of the selected carrier substance on the contents of polyphenolic compounds, antioxidant activity, color of microspheres, and ability of microspheres to inhibit α-amylase and α-glucosidase, after 14 and 28 days of storage. The results showed that appropriate selection of the polysaccharide coating is of great importance for the proper course of the microencapsulation process, the polyphenolic content of chokeberry capsules, and their antioxidant and antidiabetic properties. The addition of guar gum to a sodium alginate solution significantly increased the stability of polyphenolic compounds in microspheres during storage, whereas the addition of chitosan had a significantly negative effect on the stability of polyphenols. The coating variant composed of sodium alginate and guar gum was also found to be the most favorable for the preservation of the antioxidant activity of the capsules. On the other hand, capsules composed of sodium alginate, guar gum, and chitosan showed the best antidiabetic properties, which is related to these tricomponent microspheres having the best α-glucosidase inhibition

Sprouts vs. Microgreens as Novel Functional Foods: Variation of Nutritional and Phytochemical Profiles and Their In vitro Bioactive Properties

The aim of the study was to analyze potential health-promoting and nutritional components (polyphenols, L-ascorbic acid, carotenoids, chlorophylls, amino acids, organic acid, sugars, ash and pectins) of selected sprouts (radish, lentil, black medick, broccoli, sunflower, leek, beetroot, mung beans) and microgreens (kale, radish, beetroot, green peas, amaranth). Moreover, antioxidant capacity (2,2&prime;-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric reducing ability of plasma (FRAP), and oxygen radical absorbance capacity (ORAC)), in vitro anti-diabetic potential (inhibition of &alpha;-amylase and &alpha;-glucosidase), and anti-obesity (pancreatic lipase) and anti-cholinergic (acetylcholinesterase and butyrylcholinesterase) activity were evaluated. The results of this study show that sprouts are effective in antioxidant capacity as a result of a high content of polyphenols and L-ascorbic acid. Additionally, sprouts are better sources of amino acids, pectins and sugars than microgreens. Microgreens were characterized by high content of carotenoids and chlorophylls, and organic acid, without any sugars, exhibiting higher anti-diabetic and anti-cholinergic activity than sprouts. Some selected sprouts (broccoli, radish, lentil) and microgreens (radish, amaranths, kale) should be used daily as superfoods or functional food