MF Fiziologijos ir farmakologijos institutas (04.04)

Background: Abnormal circular smooth muscle contractility is considered a key factor in the pathogenesis of diverticular disease (DD), contributing to increased intraluminal pressure and diverticulum formation. Altered cholinergic responsiveness of colonic smooth muscle has previously been reported in DD. Although angiotensin II (Ang II) regulates gastrointestinal smooth muscle contraction through angiotensin type 1 (AT1R) and type 2 (AT2R) receptors, its role in DD remains poorly understood. The aim of the present study was to investigate alterations in contractile responses to Ang II and cholinergic stimulation in the circular muscle of the colon in DD. Methods: Colonic tissue was obtained from 16 patients with DD and 16 control patients undergoing colorectal surgery. Immunofluorescence was performed to determine the localization of AT1R and AT2R. Ang II levels in colonic muscular tissue were quantified by enzyme-linked immunosorbent assay (ELISA), AT1R and AT2R protein expression were evaluated by Western blotting, and their mRNA expression was assessed by RT-qPCR. Circular smooth muscle strips were subsequently studied using in vitro organ bath recordings to assess Ang II-induced contractile responses under basal conditions and following pharmacological blockade with losartan, tetrodotoxin (TTX), or the AT2R antagonist PD123319. Results: AT1R was predominantly localized in colonic smooth muscle cells, whereas AT2R was mainly detected in the myenteric plexus and nerve fibers. Compared with controls, DD tissues exhibited increased local Ang II levels and higher AT2R protein expression, accompanied by increased AT2R mRNA expression. Bethanechol-induced contractility was reduced in DD, whereas both peak and mean 5-min Ang II-induced contractile responses were enhanced. Losartan significantly reduced the mean 5-min Ang II-induced contractile response in both groups. However, both peak and mean responses remained significantly greater in DD than in controls following AT1R blockade. Neither TTX nor PD123319 significantly affected Ang II-induced contractions. Conclusions: The present study demonstrates altered colonic circular smooth muscle contractility in diverticular disease, characterized by enhanced responses to Ang II and reduced responses to cholinergic stimulation. The distinct muscular and neural distribution of AT1R and AT2R, respectively, suggests that altered local Ang II signaling, together with altered cholinergic regulation, may contribute to the pathogenesis of DD.

Increasing evidence suggests that mitochondria–endoplasmic reticulum contact sites (MERCs) regulate Ca²⁺ transfer, mitochondrial bioenergetics, apoptosis, and stress signalling in cardiac cells. Dysregulation of MERCs has been implicated in pathological cardiac remodelling and heart failure. These findings suggest that modulation of MERCs may represent a promising therapeutic strategy for improving cardiac function in various cardiovascular diseases. Aim: This study aims to experimentally modulate MERCs in H9c2 rat cardiac cells and assess the resulting changes in mitochondria (MIT) and endoplasmic reticulum (ER) contact-site structure and resting membrane potential. H9c2, a subclone of the original clonal cell line derived from embryonic BD1X rat (Rattus norvegicus) heart tissue was used for this study. Cultured H9c2 rat cardiac cells were transfected with ER– Mitochondrial Linkers (EMLs), molecular tools designed to maintain defined distances between ER and mitochondria [1]. Each linker includes a monomeric red fluorescent protein (mRFP) and transfection efficiency was evaluated using fluorescence microscopy. The 3D structure of MIT–ER contact sites was reconstructed using STED microscopy. The whole-cell configuration of the perforated patch clamp technique was used to record resting membrane potential in H9c2 cardiac cells. Lipofectamine 2000 and jetOPTIMUS were tested for transfection of H9c2 cells under suspension and adherent-cell conditions. Both reagents showed comparable performance, with transfection efficiencies of approximately 25%. Similar efficiencies were obtained in suspension and adherent cells, indicating no marked advantage of either reagent or transfection approach under the tested conditions. Two different linkers were used to maintain MIT-ER contacts at defined distances of 10 and 20 nm (denominated as 10nm-EML and 20nm-EML, respectively), results were compared with cells expressing control ER-mRFP plasmid (mRFP targeted to the cytosolic face of the ER). Subsequent whole-cell patchclamp recordings revealed a resting membrane potential of approximately −5.7 mV in 20nm-EML-expressing cells, compared with approximately −4.8 mV in control cells expressing ER-mRFP. Pilot experiments in cells expressing the 10 nm linker did not yield reliable electrophysiological data because stable patch-clamp recordings were technically difficult to obtain in these cells. The 3D structure of MIT-ER contact sites was created using STED microscopy images with Sir-Mito staining to visualize the spatial organization of mitochondrial–ER interactions. Both Lipofectamine 2000 and jetOPTIMUS provided comparable transfection efficiency in H9c2 cells, irrespective of whether transfection was performed in suspension or adherent conditions. STED microscopy combined with Sir-Mito staining enabled 3D reconstruction and visualization of MIT–ER contact sites. Overall, these results establish the methodological basis for further functional and structural studies of distance dependent MIT-ER interactions in cardiac cells.

Hypoxia followed by reoxygenation is associated with increased production of reactive oxygen species (ROS), which can disturb endothelial homeostasis and contribute to vascular injury. Mitochondria are an important source of ROS during reoxygenation and therefore represent a potential target for limiting oxidative damage. Hydrogen sulfide (H₂S) is an endogenous gaseous signaling molecule involved in vascular regulation and cellular redox balance. The slow-releasing H₂S donor GYY4137 has been shown to interact with mitochondrial pathways in endothelial cells [1]. However, reducing mitochondrial oxidative stress may not necessarily be sufficient for the recovery of endothelial function after hypoxic injury. This study investigated whether GYY4137 reduces mitochondrial oxidative stress and improves angiogenic function in endothelial cells following hypoxia/reoxygenation. Human umbilical vein endothelial cells (HUVECs) were exposed to 24 h hypoxia (2% O₂) followed by 24 h reoxygenation. GYY4137 (100 µM) was administered at the beginning of reoxygenation. Mitochondrial ROS production was assessed using MitoSOX Red fluorescence and normalized to MitoTracker fluorescence. Expression of CTH, encoding an H₂S-producing enzyme, and the stress-responsive gene HMOX1 was determined by quantitative real-time PCR. Endothelial angiogenic function was evaluated using a Matrigel tube formation assay, with network formation assessed after 4 and 6 h. Exposure to hypoxia increased mitochondrial ROS compared with normoxic cells, and ROS remained elevated after 24 h reoxygenation. GYY4137 treatment during reoxygenation significantly reduced mitochondrial ROS, indicating an improvement in mitochondrial redox balance. GYY4137 also increased CTH and HMOX1 expression compared with untreated hypoxic and reoxygenated cells. In contrast to these molecular changes, angiogenic function did not show a similar recovery. Hypoxia/reoxygenation impaired endothelial network formation, and GYY4137 did not significantly restore tube formation. Differences observed at the earlier time point were no longer apparent after 6 h, indicating that the effect on angiogenic network formation was limited. GYY4137 reduced persistent mitochondrial oxidative stress following hypoxia/reoxygenation and increased the expression of CTH and HMOX1. However, these changes were not accompanied by restoration of endothelial tube formation. The results suggest that recovery of mitochondrial redox balance alone is insufficient to restore angiogenic function after hypoxic injury and that additional mechanisms may limit endothelial functional recovery during reoxygenation.

Background. EVs mediate intercellular communication, with effects that may be beneficial or detrimental depending on origin and inflammatory context. This Yin-Yang concept is illustrated by two settings: therapeutic EVs from human placental stromal cells (hPSCs), expected to attenuate inflammation, and endogenous EVs in endometriosis, whose cargo may contribute to immune dysregulation.

Objectives. Using endometriosis and inflammatory lung disease as examples, we aimed to characterize EV signatures linked to immunoregulation and evaluate their immunoregulatory potential.

Methods. EVs from peritoneal fluid and plasma of 10 endometriosis patients and 16 controls were analyzed by TMT proteomics and pathway analysis. hPSC-EVs produced in 2D and 3D bioreactors were tested in PBMC assays and human 3D cystic fibrosis and ARDS airway models.

Results. Endometriosis EVs contained 2,600 plasma and 3,571 peritoneal-fluid proteins, including 251 and 285 differentially expressed proteins, with 24 shared. Altered proteins mapped to innate/inflammatory signaling, oxidative stress/iron metabolism, ECM remodeling, migration and angiogenesis - consistent with immune dysregulation and pro-inflammatory activity. In contrast, hPSC-EVs showed context-and donor-dependent anti-inflammatory activity, including macrophage IL-10 and Arg1 induction up to 25-fold.

Conclusions. These examples illustrate the Yin-Yang of EV biology: disease-associated EVs may reinforce inflammatory dysregulation, whereas stem/stromal-cell EVs can suppress inflammation, however not simply recapitulating parental cell function. EV cargo and potency therefore require context-dependent interpretation.

Introduction. Timely prevention of chronic kidney disease (CKD) and fibrosis is critical, as ~ 15% of acute kidney injury (AKI) cases progress to CKD. Identifying reliable biomarkers to assess AKI-to- CKD progression and developing preventive strategies are therefore essential. Human placental stem cells (hpMSCs), with low immunogenicity and abundant availability, are a promising source. Objectives. This study evaluated the sensitivity and correlation of AKI and CKD biomarkers, as well as the preventive effects of hpMSCs on kidney function and fibrosis. Methods. In a rat ischaemia-reperfusion injury (IRI) model, both renal pedicles were clamped for 60 minutes. The treatment group received 3 x 105 hpMSCs per kidney, while the control groups received a phosphate buffer solution (IRI-PBS), underwent IRI only, or received no intervention (healthy control). hpMSCs were isolated, cultured, and characterised for yield, viability, phenotype, and immunosuppressive capacity. Blood (creatinine), urine (KIM-1, cystatin C, NG AL) and kidney samples (acute tubular necrosis (ATN), tubular dilatation (TD), casts, loss of brush border (LBB), interstitial fibrosis and tubular atrophy (IFTA), with injury quantified by a renal injury score (RIS)) were collected on days 0, 3, 7, 14, 21-28 and 56 (n > 3 per time point; total n = 75). Correlations and comparative statistical analysis was performed using SPSS 17.0 and GraphPad Prism 7.04 with non¬parametric tests (p < 0.05) and survival curve analysis. Results. hpMSCs showed stable viability, MSC marker expression, and dose-dependent T-cell suppression. Survival reached 100% in the IRI-hpMSCs group vs. 68% (IRI-PBS, p = 0.010) and 72% (IRI, p = 0.021). Kidney function improved significantly, with lower median serum creatinine on day 7 hpMSCs vs. survived controls (IRI-hpMSCs vs. IRI, p = 0.014; IRI-PBS, p = 0.034), being ~3.5x lower than IRI- PBS and 1.6x lower than IRI. Urine cystatin C was significantly lower in the IRI-hpMSCs group on day 3 vs. IRI (p = 0.005) and IRI-PBS (p = 0.025), and urine KIM-1 was lower on day 14 vs. PBS (p = 0.02). NGAL did not differ on day 3, but was higher in the hpMSCs group on day 14 vs. IRI (p = 0.018) and PBS (p = 0.02), indicating more active tubular re-epithelialization compared to controls. Histology showed that hpMSCs reduced renal injury in both early and late phases. By day 3, ATN and casts were significantly reduced, with no focal coagulative necrosis by day 7 in the IRI - hpMSCs group. On day 14, TD, IFTA, and RIS were significantly lower in the IRI-hpMSCs vs. controls, and this reduction (TD, LBB, casts, IFTA, RIS) continued on days 21-28. IFTA remained lower on day 56 in the IRI-hpMSCs group (vs. IRI, p = 0.018; vs. IRI-PBS, p = 0.049). Serum creatinine normalized by days 28 and 56, while fibrosis persisted only in controls. Despite the limited sample size, very strong correlations (r = ±1.000; p < 0.01) were observed between urinary biomarkers (KIM-1 day 14, cystatin C day 3) and later histological IFTA scores (days 28 and 56). In the IRI-PBS group, higher KIM-1 (day 14) and cystatin C (day 3) were significantly associated with increased IFTA at day 28, compared to the IRI-hpMSCs group. A smaller decrease in median KIM-1 was still leading to increased IFTA and was inversely correlated with IFTA at day 56, suggesting possible prolonged kidney damage and delayed regeneration in the IRI-PBS group. In the IRI group, the higher cystatin C (day 3) compared to the IRI-hpMSCs group was strongly associated with increased IFTA on day 56 (p = 0.001). In the IRI-hpMSCs group bigger decrease in median KIM-1 (day 14) was leading near-zero IFTA, showing a very strong inverse correlation (p = 0.001) between still numerically higher KIM-1 (day 14) as compared to day 0. Similarly, decreased cystatin C levels on day 3 are strongly associated with lower IFTA values in the cell group on day 28 (p = 0.001). Conclusion. We demonstrated the potential of hPSCs to prevent the initial cascade of kidney fibrosis and death by reducing early renal injury and improving kidney function in both the acute and late phases of AKI. 'Ihe 100% survival rate in the hPSCs-treated group highlights their potential for further mechanistic, large-scale studies, and subsequent clinical trials.

Aim: In vitro studies on intestinal smooth muscles from patients with diverticular disease (DD) have demonstrated impaired relaxation, likely due to dysfunction of the nitrergic neurotransmission system[1,2]. Activation of angiotensin II type 2 receptors (AT2R) is known to stimulate nitric oxide (NO) production [3], a key mediator of colonic smooth muscle relaxation [4]. This study aims to investigate the effects of AT2R inhibition on colonic circular muscle (CCM) contractility in DD. Methods: The control samples were obtained from patients with colorectal carcinoma, while the other group samples were collected from patients with asymptomatic DD. Immunohistochemistry was performed for HuC/HuD (neuronal bodies) and AT2R. The area of neuronal cell bodies and AT2R-positive staining in myenteric plex-uses were quantified using ImageJ. CCM strips were dissected and mounted in organ baths containing warm, oxygenated Krebs solution for isometric recording of contractile activity in response to Ang II (10 -6 M), alone or in combination with the AT2R antagonist(PD123319, 10 -7 M) and tetrodotoxin (TTX, 10 -7 M). Reverse transcriptase polymerase chain reaction (RT-PCR) was used to investigate the presence of AT2R mRNA in both groups. Results: Neuronal count in myenteric ganglia was significantly reduced in DD compared with controls (17.45 and 20.23, respectively; p = 0.009). In contrast, AT2R expression was modestly increased in DD (10.51 and 9.78, respectively; p = 0.01), supported by higher AT2Rgene activity (p = 0.003). Colonic circular muscle (CCM) from DD patients exhibited enhanced contractile responses to Ang II (61.47 and43.52, respectively; p = 0.035), further augmented by AT2R antagonism (65.82 and 39.01, respectively; p = 0.04) and preserved under neural blockade (70.19 and 41.96, respectively; p = 0,045). Conclusion: DD is associated with increased AT2R expression and CCM contractility, likely due to Ang II receptor imbalance, suggesting predominant AT1 receptor–mediated effects

Background/Objectives: Hydrogen sulfide (H2S) is an important regulator of pulmonary vascular tone; however, the mechanisms underlying its biphasic contractile and relaxant effects, as well as the distinct contributions of endogenous and exogenous H2S to nitric oxide (NO) signaling, remain incompletely understood. This study investigated the roles of endogenous and exogenous H2S in regulating pulmonary vascular responses under physiological and oxidative stress conditions. Methods: Rat intrapulmonary arteries were studied using wire myography and simultaneous measurements of vascular force and intracellular Ca2+. Pharmacological inhibitors were used to examine the contribution of endogenous H2S synthesis, NO synthase, soluble guanylate cyclase (sGC), KATP channels, and oxidative stress to vascular responses. Results: Sodium hydrogen sulfide (NaHS) produced biphasic concentration-dependent responses, with contraction at low concentrations followed by relaxation at higher concentrations. Simultaneous measurements of vascular force and intracellular Ca2+ showed that the initial contractile response to low concentrations of NaHS was not accompanied by a clear increase in intracellular Ca2+, whereas relaxation developed despite maintained or increasing intracellular Ca2+, indicating that NaHS-induced relaxation cannot be explained solely by reduced intracellular Ca2+ levels. Inhibition of endogenous H2S synthesis reduced vascular responsiveness to the NO donor sodium nitroprusside, whereas relaxation induced by exogenous NaHS was largely preserved after inhibition of NO synthase or sGC, indicating distinct roles of endogenous and exogenous H2S. Oxidative stress impaired acetylcholine- and riociguat-induced relaxation, while NaHS partially restored endothelial function. Conclusions: Endogenous and exogenous H2S regulate pulmonary vascular tone through distinct mechanisms. Endogenous H2S supports vascular responsiveness to NO, whereas exogenous H2S induces relaxation largely independently of NO–sGC signaling. Furthermore, NaHS-induced relaxation occurs despite maintained intracellular Ca2+, suggesting an important contribution of Ca2+-independent mechanisms, and partially preserves endothelial function under oxidative stress.

To evaluate the methodological quality of hospital-specific paediatric sepsis clinical practice guidelines (CPGs) from sites participating in an international study using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) tool and to assess the consistency of treatment recommendations and concordance with the Surviving Sepsis Campaign 2020 (SSC 2020) international reference standard.

The development of effective delivery systems remains an important challenge in modern pharmaceutical and biomedical research, driving the development of biocompatible materials with versatile functional properties. This review provides an integrated overview of chitosan-based delivery systems, focusing on chitosan nanoparticles, chitosan derivatives, and hybrid systems. Particular emphasis is placed on the relationship between the structural and functional characteristics of chitosan-based systems and their potential applications in pharmaceutical and biomedical fields. In addition to established applications, emerging approaches involving nucleic acid delivery, gene therapy, theranostics, and other biomedical applications are discussed. A further focus of this review is the consideration of critical quality attributes relevant to the development of GMP-oriented chitosan-based systems, providing a perspective that links material characteristics with quality and translational requirements. By integrating these aspects, this review highlights the versatility of chitosan as a platform for the development of innovative pharmaceutical and biomedical delivery technologies.

Virusinės kvėpavimo takų infekcijos yra siejamos su ilgalaikiais centrinės nervų sistemos pokyčiais [1]. COVID-19 persirgusiems pacientams kartu su kognityvinių funkcijų sutrikimu yra nustatomas ir kraujo–smegenų barjero pralaidumo padidėjimas [2]. Vienas iš galimų šį ryšį paaiškinančių mechanizmų yra plaučių–smegenų ašis, per kurią kvėpavimo sistemoje susidarę signalai gali veikti kraujo–smegenų barjerą ir mikrogliją [3]. Vienas iš galimų signalų perdavėjų yra užląstelinės pūslelės (UP) – membrana dengtos nanodalelės, kurių turinys priklauso nuo jas išskiriančios ląstelės būklės [4]. Kraujo–smegenų barjere UP gali veikti smegenų kapiliarų endotelį, kurio pralaidumas priklauso nuo glaudžiųjų jungčių, sudarytų iš baltymų, tokių kaip ZO-1 ir klaudinas-5 [5]. Todėl šio tyrimo tikslas – įvertinti virusiniais veiksniais stimuliuotų kvėpavimo takų epitelio ląstelių išskirtų UP poveikį žmogaus smegenų kapiliarų endotelio ląstelių barjerinei funkcijai. Šiame tyrime naudotos komercinės žmogaus bronchų epitelio (HBEC3-KT) ir smegenų kraujagyslių endotelio (hCMEC/D3) ląstelių linijos. Virusiniam uždegimui imituoti ląstelės buvo paveiktos 0,1 mg/mL poli I:C (TLR3 receptoriaus agonistu) arba 0,1 µg/mL SARS-CoV-2 spyglio baltymo receptorių surišančiu domenu (RBD). HBEC3-KT ląstelėms pasiekus 90 % konfluentiškumą, viena jų grupė buvo 2 h inkubuojama su poli I:C, kita – 48 h su RBD, o nepaveiktos ląstelės naudotos kaip kontrolė. UP buvo išskirtos iš ląstelių kultūros terpės polimerinės precipitacijos metodu, o jų dydis bei koncentracija nustatyti nanodalelių sekimo analizės būdu. Endotelio ląstelės su išskirtomis UP buvo inkubuojamos 48 h, o siekiant palyginti UP ir tiesioginį virusinių veiksnių poveikį, endotelio ląstelės taip pat 48 h buvo veikiamos poli I:C ar RBD. UP ir virusinių veiksnių poveikis endotelio barjerinėms savybėms buvo vertinamas matuojant transendotelinę elektrinę varžą (TEER) bei imunocitochemiškai analizuojant ZO-1 ir klaudino-5 organizaciją endotelio ląstelėse, vaizdinant fluorescenciniu mikroskopu (Olympus ApexView APX100). Duomenų analizė atlikta naudojant „Microsoft Excel“ ir „ImageJ“ programas. Nanodalelių sekimo analizė parodė, kad išskirtų dalelių dydis svyravo nuo 50 nm iki 400 nm, o koncentracija nuo 1,26×1010 iki 2,30×1010 dalelių/ml. Tiek tiesioginis poli I:C ir RBD poveikis, tiek išskirtų UP poveikis mažino endotelio transendotelinę elektrinę varžą, o ryškiausias sumažėjimas nustatytas ląsteles veikiant RBD stimuliuotų epitelio ląstelių UP. Imunocitocheminė analizė parodė ZO-1 ir klaudino-5 organizacijos pokyčius ląstelių membranose, pasireiškiančius jungčių fragmentacija. Gauti rezultatai rodo, kad virusiniais veiksniais stimuliuotų kvėpavimo takų epitelio ląstelių išskirtos UP gali sutrikdyti žmogaus smegenų kapiliarų endotelio barjerines savybes, mažindamos transendotelinę elektrinę varžą bei keisdamos ZO-1 ir klaudino-5 organizaciją tarpląstelinėse jungtyse.