ROCK Activity Research Articles

ИСПОЛЬЗОВАНИЕ АКТИВИРОВАННОГО ПРИРОДНОГО ЦЕОЛИТА В КАЧЕСТВЕ АДСОРБЕНТА ДЛЯ ОЧИСТКИ ПРИРОДНЫХ ВОД ОТ ИОНОВ МЕТАЛЛОВ

The possibility of using natural zeolites as adsorbents for water treatment is shown. For experimental studies of sorption properties of natural zeolite was used zeolite-containing rock of the Tatarsko-Shatrashansky deposit, with a fraction size less than 1 mm. The structure, physical and chemical properties of this zeolite are considered. Features of different methods of zeolite activation are given. Acid treatment is an effective way of activation, which has a significant effect on the chemical composition and structure of natural minerals. The comparative analysis of acid and alkaline activation of zeolite is carried out, the advantages of acid treatment are revealed. A simplified method of chemical activation of natural zeolite is proposed, which allows to increase its adsorption capacity. The results of acid activation of zeolite-containing rock of the Tatarsko-Shatrashansky deposit are presented. Sorption characteristics of zeolite-containing sorbent allow its use for removal of metal ions from waters of different origin. Model solutions containing Cu(II), Mn(II), Cr(VI), Zn(II) ions were prepared for analysis. The dynamics of metal ions sorption on zeolite was evaluated. It was found that zeolite shows the most effective sorption properties towards Zn(II), Cr(VI) and Cu(II) ions. Sorption efficiency within 60 minutes for zinc ions reaches about 45% for chromium ions -58%, for copper ions - 80%. The obtained results on the use of this activated zeolite for purification of water from metal ions also allow us to consider the possibility of using this zeolite for purification of wastewater used secondary for preparation of process solutions in galvanic production.

Proteomic Profiling and Cytokine Analysis Reveals Molecular Signatures and Inflammatory Factors Associated with Thrombosis in JAK2-V617F Positive Myeloproliferative Neoplasm (MPN) Cohorts

Background: Myeloproliferative neoplasms (MPNs), a hematological malignancy commonly associated with the JAK2-V617F mutation, exhibit a considerable burden of comorbidities resulting from inflammation-mediated processes. The JAK2-V617F malignant clones instigate systemic inflammation by upregulating various pro-inflammatory cytokines. This leads to dysregulated immune and inflammatory responses, thereby driving a process called thrombo-inflammation. Neutrophils play a crucial role as inflammatory mediators and effectors of pro-thrombotic activity in MPNs. We, therefore, hypothesize that perturbations in the neutrophil proteome may alter the molecular communication networks within neutrophils, thereby potentially contributing to the pathological thrombus formation in veins and arteries. Aim: The study was aimed to elucidate (i) the pathological significance of dysregulated protein expression profile of neutrophils carrying the JAK2-V617F mutation, and (ii) the impact of pro-inflammatory cytokines on neutrophils during thrombo-inflammation. Methods: In this study, which was conducted at the University Hospital Magdeburg, Germany, 11 patients carrying the JAK2-V617F mutation (6 with thrombosis; 5 without thrombosis) and 9 healthy volunteers were enrolled. Neutrophils were isolated from freshly collected whole blood using a negative selection kit. The isolated neutrophils were then lysed, and protein extraction was performed. High-resolution liquid chromatography - mass spectrometry (LC-MS/MS) was employed to investigate the protein inventory of neutrophils. Furthermore, platelet-poor plasma was isolated from the same cohort to analyse plasma cytokine/chemokine levels using a bead-based multiplex assay. Results: LC-MS/MS identified 2,006 proteins in neutrophils, which were then used for quantitative comparative analysis. Amongst these, 28 and 26 proteins exhibited significant up-regulation in the JAK2-V617F positive patients ‘with thrombosis’ and ‘without thrombosis’, respectively. Notably, ATP2A3 and ROCK1 were significantly upregulated in the JAK2-V617F thrombotic group, ranking among the top 10-upregulated proteins. Of note, ROCK1 activation has previously been implicated in cell proliferation, survival and cell cycle control. Functional annotation analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that upregulated proteins in the JAK2-V617F cohorts were significantly enriched for proteins involved in the calcium and TGF-ß signaling pathways. Furthermore, we assessed the levels of 60 cytokines/chemokines and soluble adhesion molecules in the same cohort, wherein significantly increased levels of ALCAM, ICAM-2 and VCAM-1 were observed in the JAK2-V617F thrombotic group. These molecules are known to play a critical role in vasculature-related inflammation and endothelial cell activation. Additionally, the JAK2-V617F thrombotic group exhibited significant upregulation of free active TGF-ß and pro-inflammatory cytokines such as IL-6 and IL-8 compared to both healthy subjects and JAK2-V617F non-thrombotic patients, indicating their contribution to the thrombo-inflammatory response. Moreover, the thrombotic cohort displayed elevated plasma levels of CXC chemokines and myeloperoxidase (MPO), a key enzyme linked to NETosis for neutrophil extracellular trap (NET) formation. Conclusions: The proteomic profile of neutrophils in MPN patients exhibit significant alterations, particularly between JAK2-V617F patients with and without thrombosis. The observed elevated expression of markers related to NETosis and calcium signaling pathways point towards the underlying mechanisms that could potentially contribute to the differential behavior of neutrophils, thereby facilitating the development of a thrombotic state in MPN patients.

Urinary exosomes from patients with diabetic kidney disease induced podocyte apoptosis via microRNA-145-5p/Srgap2 and the RhoA/ROCK pathway

Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease without early diagnostic and specific therapeutic approaches. Podocyte apoptosis and loss play important roles in the pathological process of DKD. This study aimed to explore whether urinary exosomes from type 2 diabetes patients with DKD could induce podocyte apoptosis and the underlying pathological mechanisms. The exosomes were isolated from the urine samples of patients with DKD (DKD-Exo). Later, they were taken up and internalized by MPC5 cells. MPC5 cells were co-cultured with DKD-Exo (45 μg/ml) for 24 h in the presence or absence of microRNA-145-5p (miR-145-5p) inhibitor, fasudil and pcDNA-Srgap2 transfection. MiR-145-5p and Srgap2 expression was evaluated using real-time quantitative PCR. The protein levels of Srgap2, Bcl-2, Bax, and cleaved caspase-3, as well as ROCK activity were determined using Western blotting. Cell apoptosis was measured using flow cytometry and the TUNEL assay. miR-145-5p expression in MPC5 cells exposed to DKD-Exo was markedly upregulated. miR-145-5p negatively regulated Srgap2 levels. Exposure of MPC5 cells to DKD-Exo reduced Srgap2 expression and activated ROCK, which was partly reversed by the presence of the miR-145-5p inhibitor or Srgap2 overexpression. The apoptosis of MPC5 cells exposed to DKD-Exo increased significantly, which was counteracted by the addition of the miR-145-5p inhibitor and fasudil. The results showed that urinary exosomal miR-145-5p from patients with DKD induced podocyte apoptosis by inhibiting Srgap2 and activating the RhoA/ROCK pathway, suggesting that urinary exosomal miR-145-5p is involved in the pathological process of DKD and could become a noninvasive diagnostic biomarker for DKD.

Shotgun metagenomics from Monte Cristo cave (Brazil) reveals microbial metabolic potential related to iron and manganese biogeochemical cycles

Caves are among the most singular and understudied environments on Earth. Due to the harsh conditions observed in many caves, including scarcity of nutrients and low levels of light, these ecosystems are considered extreme environments (Gabriel and Northup 2012). Therefore, it may be worth paying special attention to the microbial communities existing in these unique systems. Previously, it has been suggested that the high levels of Manganese (Mn) and Iron (Fe) at Lechuguilla and Spider Caves in the United States may influence their microbial community structure in different ways (Northup et al. 2003, Carmichael and Bräuer 2015). In this context, caves are promising environments for investigating microbial functional capabilities in relation to these elements and the ecological interactions that allow these microbes to thrive. Monte Cristo cave (MCC) - in Diamantina, Brazil - was chosen for this study. The cave is located in a region known for historic mining activity and occurrences of Mn and Fe-rich rocks (Costa et al. 2003). With that in mind, our main goal is to investigate if within the microbial community of MCC there is evidence of taxa and genes associated with Fe and Mn metabolism. The samples were collected in 2018 from walls and saprolite deposits within MCC. Community DNA from three samples, P1b, P3 and P7, were independently sequenced using Illumina shotgun sequencing, and the data were analysed using conventional metagenomic pipelines and in-house python scripts. Taxonomic classification was assessed using Kraken2; Fe related genes with FeGenie; and Mn related genes were predicted using BlastP against a collection of manually curated Mn-oxidizing proteins. Environmental Mn and Fe concentrations were measured using ICP-OES. Our results suggest the presence of a microbial community potentially able to change Fe and Mn redox states. In sites P1b and P7, genes associated to Fe and Mn oxidation were identified, Fig. 1. Taxonomic evidence for these metabolisms includes the presence of the taxa Comamonadaceae and Hyphomicrobiaceae, both families that were previously reported to harbour species able to oxidize Mn and Fe (Spring and Kämpfer 2015, Carmichael and Bräuer 2015). Our analysis also assigned contigs to the archaeal phyla Crenarchaeota, Euryarchaeota and Thaumarchaeota, whose presence has been associated with oligotrophic caves where archaea play a role in primary production (Ortiz et al. 2013) (Fig. 2). Moreover, the phylum Euryarchaeota harbours members that use Fe or Mn as electron acceptors during methane oxidation (Ettwig et al. 2016). Our results therefore contribute to understanding how microbial communities of MCC may be playing a role in the biogeochemical cycles of Fe and Mn under the conditions imposed by the subterranean environment, which might reflect similar processes in other caves yet to be explored by a metagenomics approach.

Geochemistry and petrology of the Chandarpur-Raipur sandstones: Implications for the evolution of the Chattisgarh Basin, Central India

Depositional processes and stratigraphic evolution of the Chattisgarh basin exhibit a synergistic relation between sedimentation and tectonics. To interpret the sedimentation history and basin evolution, multiproxy approach including field studies, petrography, geochemical, and multivariate statistical analyses of the sandstones of Chandarpur and Raipur groups is employed. The study provides a detailed analysis of the time-connoted petrography and geochemistry of the sandstone units from each formation of the Chandarpur-Raipur sequence of Chattisgarh basin. The sandstones grade from immature wacke through submature arkose to mature quartzarenite from the bottom up the section. These sandstones are broadly characterized by felsic provenance, arid-humid palaeoclimate, and weak-intense palaeoweathering at the source. The assemblage of wacke-arkose-sublitharenite of the basal Lohardih Formation is characteristics of fan delta environment and represents initiation of rifting and basin formation stage. Subarkoses from the younging up sequence of Lohardih-Chaporadih-Kansapathar formations reflect prograding shelf environment that represents craton interior field of the continental block provenance and passive marginal tectonic setting, indicating gradual stabilization of the basin. Quartzarenites from Kansapathar Formation and stratigraphically younger Devsagar Member of Charmuria Formation also represent broadly similar provenance and tectonic setting indicating stable craton interior provenance in a passive marginal setting. Petrographic and geochemical analyses of some of the Kansapathar-Devsagar sandstones suggesting recycled orogen provenance, however, may be attributed to the orogenic activity and/or role of younger bimodal volcanic rock during rejuvenation of multistage basin opening faults. Rifting stage was followed by subsidence stage as indicated by the presence of black limestone capping the sandstone successions. Initiation of basin formation ∼1400 Ma and sedimentation can be correlated with the late-stage fragmentation of the supercontinent Columbia. The basin probably closed ∼1000 Ma during the assembly of the next supercontinent Rodinia. It is estimated that ∼87% of the 2.2-km-thick sedimentary succession in the eastern Chattisgarh basin, were already deposited within this time frame (∼1405 and ∼1007 Ma). The basin is correlatable with other Mesoproterozoic basins of Peninsular India like Vindhyan, Cuddapah basins, as well as with basins like Xiong’er rift basin of North China Craton, Espinhaco basin of eastern Brazil, that are related to the Columbia-Rodinia Supercontinent cycle.

Selective inhibition of cannabinoid CB1 receptor-evoked signalling by the interacting protein GAP43

Cannabinoids exert pleiotropic effects on the brain by engaging the cannabinoid CB1 receptor (CB1R), a presynaptic metabotropic receptor that regulates key neuronal functions in a highly context-dependent manner. We have previously shown that CB1R interacts with growth-associated protein of 43 kDa (GAP43) and that this interaction inhibits CB1R function on hippocampal excitatory synaptic transmission, thereby impairing the therapeutic effect of cannabinoids on epileptic seizures in vivo. However, the underlying molecular features of this interaction remain unexplored. Here, we conducted mechanistic experiments on HEK293T cells co-expressing CB1R and GAP43 and show that GAP43 modulates CB1R signalling in a strikingly selective manner. Specifically, GAP43 did not affect the archetypical agonist-evoked (i) CB1R/Gi/o protein-coupled signalling pathways, such as cAMP/PKA and ERK, or (ii) CB1R internalization and intracellular trafficking. In contrast, GAP43 blocked an alternative agonist-evoked CB1R-mediated activation of the cytoskeleton-associated ROCK signalling pathway, which relied on the GAP43-mediated impairment of CB1R/Gq/11 protein coupling. GAP43 also abrogated CB1R-mediated ROCK activation in mouse hippocampal neurons, and this process led in turn to a blockade of cannabinoid-evoked neurite collapse. An NMR-based characterization of the CB1R-GAP43 interaction supported that GAP43 binds directly and specifically through multiple amino acid stretches to the C-terminal domain of the receptor. Taken together, our findings unveil a CB1R-Gq/11-ROCK signalling axis that is selectively impaired by GAP43 and may ultimately control neurite outgrowth.

Y-27632 acts beyond ROCK inhibition to maintain epidermal stem-like cells in culture.

Conditional reprogramming is a cell culture technique that effectively immortalizes epithelial cells with normal genotypes by renewing epidermal stem cells. Y-27632, a compound that promotes conditional reprogramming through an unknown mechanism, was developed to inhibit the two Rho-associated kinase (ROCK) isoforms. We used human foreskin keratinocytes (HFKs) to study the role of Y-27632 in conditional reprogramming and learn how ROCKs control epidermal stem cell renewal. In conditional reprogramming, Y-27632 increased HFK adherence to culture dishes, progression through S, G2 and M phases of the cell cycle, and epidermal stem cell marker levels. Although this correlated with ROCK inhibition by Y-27632, we generated CRISPR-Cas9-mediated HFK ROCK knockouts to test the direct role of ROCK inhibition. Knockout of single ROCK isoforms was insufficient to disrupt ROCK activity or promote HFK propagation without Y-27632. Although ROCK activity was reduced, HFKs with double knockout of ROCK1 and ROCK2 still required Y-27632 to propagate. Y-27632 was the most effective among the ROCK inhibitors we tested at promoting HFK proliferation and epidermal stem cell marker expression. Thus, the ability of Y-27632 to promote an epidermal stem cell state in conditional reprogramming not only depends upon ROCK inhibition but also acts via as-yet-unidentified mechanisms. Epidermal stem cell renewal might in part be regulated by ROCKs, but also involves additional pathways.

Migration and differentiation of muscle stem cells are coupled by RhoA signalling during regeneration.

Skeletal muscle is highly regenerative and is mediated by a population of migratory adult muscle stem cells (muSCs). Effective muscle regeneration requires a spatio-temporally regulated response of the muSC population to generate sufficient muscle progenitor cells that then differentiate at the appropriate time. The relationship between muSC migration and cell fate is poorly understood and it is not clear how forces experienced by migrating cells affect cell behaviour. We have used zebrafish to understand the relationship between muSC cell adhesion, behaviour and fate in vivo. Imaging of pax7-expressing muSCs as they respond to focal injuries in trunk muscle reveals that they migrate by protrusive-based means. By carefully characterizing their behaviour in response to injury we find that they employ an adhesion-dependent mode of migration that is regulated by the RhoA kinase ROCK. Impaired ROCK activity results in reduced expression of cell cycle genes and increased differentiation in regenerating muscle. This correlates with changes to focal adhesion dynamics and migration, revealing that ROCK inhibition alters the interaction of muSCs to their local environment. We propose that muSC migration and differentiation are coupled processes that respond to changes in force from the environment mediated by RhoA signalling.

STRESS ANALYSIS AND CHARACTERISTICS DUE TO THE SOUTH JAVA EARTHQUAKE, APRIL 10, 2021

The April 10, 2021, earthquake in the south of East Java was classified as destructive. The secondary impact of this earthquake was quite significant. Many houses collapsed, and not a few casualties. This earthquake is unique because usually, destructive earthquakes occur at shallow depths, but earthquakes with a magnitude of 6.1 are classified as medium-depth earthquakes at sea. The earthquake in the south of East Java is classified as an intraplate earthquake because it is located on the continental plate, not in the plate contact area. The question is whether the damage that occurred to the building was purely due to the magnitude of the stress released by the earthquake or whether there were other factors. This study uses seismogram data for the earthquake south of East Java on April 10, 2021, with a radius (∆) of 300-1000 recorded at MEEK, MORW, and ARMA stations in Australia. It calculates the amount of stress based on the stress drop, while the stress column determines the stress mechanism. Calculation of stress drop from the source spectrum is obtained by the deconvolution method, namely the seismogram component separation technique in the form of Source (f), Path (f), Site (f), and Instrument (f). The analysis of the observed displacement spectrum used the Nelder Mead Simplex nonlinear inversion method. Meanwhile, the Stress Columb calculation was obtained using the Columb 3.3 program from the United States Geological Survey (USGS). The result of this research is that the stress drop value is 1.69 MPa, with the type of focus mechanism being a thrust fault in the sea. The earthquake in the south of East Java was caused by rock activity in the intraplate. The value of the stress drop is more significant when compared to the subduction contact area. This area is of intraplate rock with various variations, and earthquakes are rare. This study aims to analyze the stress, both the magnitude of the stress drop and the mechanism of the column stress results, so that the stress caused by the earthquake can be known and why the earthquake in the south of East Java is destructive. The quake in Southeast Java is classified as dangerous, not because of the magnitude of the stress generated or its mechanism. The damage was due to the amplification of earthquake waves in the building. The injury occurred because most of the buildings were built on soft soil, especially in several areas in East Java, such as Lumajang, Pasuruan, Trenggalek, Probolinggo, Ponorogo, Jember, Tulunggagung, Nganjuk, Pacitan, and several urban areas, namely Blitar, Kediri, Malang, and Stone. So, there is a need for earthquake disaster mitigation, especially in densely populated areas that live on soft soil. This mitigation effort is to minimize the occurrence of casualties by building buildings according to earthquake-resistant standards and avoiding development in the regions that have the potential for amplification of earthquake waves.

Catalytic Conversion of Oil in Model and Natural Reservoir Rocks

The catalytic activity of metal oxides in the processes of low- and high-temperature oxidation (LTO and HTO, respectively) of oil was studied on model systems consisting of oil-saturated quartz sand with additives of Al2O3, Cr2O3 and MgO using thermal analysis methods. The used additives were shown to shift the LTO and HTO processes to the low-temperature region. The catalytic activity of a natural reservoir carbonate rock without and with water was studied. This study established that at room temperature in dry carbonate rock, the oil components undergo dealkylation and polycondensation of aromatic fragments for a week. In the presence of water, the polycondensation processes are suppressed, and the cracking of resin and asphaltene occurs. The cracking reactions lead to a decrease in the content of heteroatoms in resins and asphaltenes.

Peristalsis-Associated Mechanotransduction Drives Malignant Progression of Colorectal Cancer.

In the colorectal cancer (CRC) tumor microenvironment, cancerous and precancerous cells continuously experience mechanical forces associated with peristalsis. Given that mechanical forces like shear stress and strain can positively impact cancer progression, we explored the hypothesis that peristalsis may also contribute to malignant progression in CRC. We defined malignant progression as enrichment of cancer stem cells and the acquisition of invasive behaviors, both vital to CRC progression. We leveraged our peristalsis bioreactor to expose CRC cell lines (HCT116), patient-derived xenograft (PDX1,2) lines, or non-cancerous intestinal cells (HIEC-6) to forces associated with peristalsis in vitro. Cells were maintained in static control conditions or exposed to peristalsis for 24 h prior to assessment of cancer stem cell (CSC) emergence or the acquisition of invasive phenotypes. Exposure of HCT116 cells to peristalsis significantly increased the emergence of LGR5+ CSCs by 1.8-fold compared to static controls. Peristalsis enriched LGR5 positivity in several CRC cell lines, notably significant in KRAS mutant lines. In contrast, peristalsis failed to increase LGR5+ in non-cancerous intestinal cells, HIEC-6. LGR5+ emergence downstream of peristalsis was dependent on ROCK and Wnt activity, and not YAP1 activation. Additionally, HCT116 cells adopted invasive morphologies when exposed to peristalsis, with increased filopodia density and epithelial to mesenchymal gene expression, in a Wnt dependent manner. Peristalsis associated forces drive malignant progression of CRC via ROCK, YAP1, and Wnt-related mechanotransduction. The online version contains supplementary material available at 10.1007/s12195-023-00776-w.

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress, mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway.

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1S616 , mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.

SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

Allergic asthma is characterized by goblet cell metaplasia and subsequent mucus hypersecretion that contribute to the morbidity and mortality of this disease. Here, we explore the potential role and underlying mechanism of protein SUMOylation-mediated goblet cell metaplasia. The components of SUMOylaion machinery are specifically expressed in healthy human bronchial epithelia and robustly upregulated in bronchial epithelia of patients or mouse models with allergic asthma. Intratracheal suppression of SUMOylation by 2-D08 robustly attenuates not only allergen-induced airway inflammation, goblet cell metaplasia, and hyperreactivity, but IL-13-induced goblet cell metaplasia. Phosphoproteomics and biochemical analyses reveal SUMOylation on K1007 activates ROCK2, a master regulator of goblet cell metaplasia, by facilitating its binding to and activation by RhoA, and an E3 ligase PIAS1 is responsible for SUMOylation on K1007. As a result, knockdown of PIAS1 in bronchial epithelia inactivates ROCK2 to attenuate IL-13-induced goblet cell metaplasia, and bronchial epithelial knock-in of ROCK2(K1007R) consistently inactivates ROCK2 to alleviate not only allergen-induced airway inflammation, goblet cell metaplasia, and hyperreactivity, but IL-13-induced goblet cell metaplasia. Together, SUMOylation-mediated ROCK2 activation is an integral component of Rho/ROCK signaling in regulating the pathological conditions of asthma and thus SUMOylation is an additional target for the therapeutic intervention of this disease.

Evaluation of the pulmonary toxicity of PSNPs using a Transwell-based normal human bronchial epithelial cell culture system

To reduce the nanoplastics (NPs) toxicity assessment error, we established a Transwell-based bronchial epithelial cell exposure system to assess the pulmonary toxicity of polystyrene NPs (PSNPs). Transwell exposure system was more sensitive than submerged culture for toxicity detection of PSNPs. PSNPs adhered to the BEAS-2B cell surface, were ingested by the cell, and accumulated in the cytoplasm. PSNPs induced oxidative stress and inhibited cell growth through apoptosis and autophagy. A noncytotoxic dose of PSNPs (1 ng/cm2) increased the expression levels of inflammatory factors (ROCK-1, NF-κB, NLRP3, ICAM-1, etc) in BEAS-2B cells, whereas a cytotoxic dose (1000 ng/cm2) induced apoptosis and autophagy, which might inhibit the activation of ROCK-1 and contribute to reducing inflammation. In addition, the noncytotoxic dose increased the expression levels of zonula occludens-2 (ZO-2) and α1-antitrypsin (α-AT) proteins in BEAS-2B cells. Therefore, in response to PSNP exposure, a compensatory increase in the activities of inflammatory factors, ZO-2, and α-AT may be triggered at low doses as a mechanism to preserve the survival of BEAS-2B cells. In contrast, exposure to a high dose of PSNPs elicits a noncompensatory response in BEAS-2B cells. Overall, these findings suggest that PSNPs may be harmful to human pulmonary health even at an ultralow concentration.

USF2 activates RhoB/ROCK pathway by transcriptional inhibition of miR-206 to promote pyroptosis in septic cardiomyocytes.

Septic cardiomyopathy (SCM) is one of the most serious complications of sepsis. The present study investigated the role and mechanism of upstream stimulatory factor 2 (USF2) in SCM. Serum samples were extracted from SCM patients and healthy individuals. A murine model of sepsis was induced by caecal ligation and puncture (CLP) surgery. Myocardial injury was examined by echocardiography and HE staining. ELISA assay evaluated myocardial markers (CK-MB, cTnI) and inflammatory cytokines (TNF-α, IL-1β, IL-18). Primary mouse cardiomyocytes were treated with lipopolysaccharide (LPS) to simulate sepsis in vitro. RT-qPCR and Western blot were used for analyzing gene and protein levels. CCK-8 assay assessed cell viability. NLRP3 was detected by immunofluorescence. ChIP, RIP and dual luciferase reporter assays were conducted to validate the molecular associations. USF2 was increased in serum from SCM patients, septic mice and primary cardiomyocytes. USF2 silencing improved the survival of septic mice and attenuated sepsis-induced myocardial pyroptosis and inflammation in vitro and in vivo. Mechanistically, USF2 could directly bind to the promoter of miR-206 to transcriptionally inhibit its expression. Moreover, RhoB was confirmed as a target of miR-206 and could promote ROCK activation and NLRP3 inflammasome formation. Moreover, overexpression of RhoB remarkably reversed the protection against LPS-induced inflammation and pyroptosis mediated by USF2 deletion or miR-206 overexpression in cardiomyocytes. The above findings elucidated that USF2 knockdown exerted a cardioprotective effect on sepsis by decreasing pyroptosis and inflammation via miR-206/RhoB/ROCK pathway, suggesting that USF2 may be a novel drug target in SCM.

405-P: ROCK2 Activation and Its Role in Folic Acid–Induced Murine Model of Nephropathy

Over 2 million people currently receive treatment with dialysis or a kidney transplant throughout the world. Thus, developing a new strategy to treat chronic kidney diseases is significant from the perspective of a health economic and the prevention of cardiovascular death. The small GTPase Rho and its effector Rho-kinase (ROCK) are involved in the pathogenesis of diabetic nephropathy. In chronic kidney diseases including diabetic nephropathy, tubulointerstitial fibrosis is known to be highly associated with renal outcome. We have previously reported that ROCK inhibition prevented the progression of diabetic nephropathy in murine models. With regards to ROCK, there are two isoforms, which are ROCK1 and ROCK2. ROCK1 has been shown to be involved in the endocytosis of albumin in tubular epithelial cells via megalin/cubilin-dependent mechanism. However, the role of renal tubular ROCK2 remains to be elucidated. The aim of this study is to reveal the role of renal tubular ROCK2 in the pathogenesis of chronic kidney disease. To this end, we used a tubulointerstitial injury model known as folic acid-induced nephropathy (FAN). Importantly, ROCK2 was elevated at protein levels in FAN group compared with vehicle-treated group. We next generated tubular specific ROCK2 knockout mouse (TR2KO) by crossing Ksp-Cre mice with mice containing two loxP sites flanking in Exon 3 of ROCK2. In the Ksp-Cre transgenic mice, Cre recombinase has been demonstrated to be expressed in tubules, collecting ducts, and thick ascending limbs. Compared with vehicle-treated group, collagen deposition in the kidneys of FAN group was highly elevated as revealed by Masson’s trichrome staining. The increased collagen deposition was significantly improved in TR2KO. Our findings indicate that ROCK2 regulates the progression of tubulointerstitial fibrosis. ROCK2 may be a potential therapeutic target for the prevention of chronic kidney disease. Disclosure K.Sekiguchi: None. K.Matoba: None. S.Ohashi: None. E.Mitsuyoshi: None. Y.Nagai: None. Y.Takeda: None. R.Nishimura: Speaker's Bureau; Sanofi K.K., Medicure Inc., Boehringer Ingelheim Inc., Takeda Pharmaceutical Company Limited, Kissei Pharmaceutical Co., Ltd., Novartis Pharma K.K., Eli Lilly Japan K.K., Novo Nordisk, Merck & Co., Inc., Abbott Japan Co., Ltd., Astellas Pharma Inc., Teijin Pharma Limited, Taisho Pharmaceutical Holdings Co., Ltd., Ono Pharmaceutical Co., Ltd., AstraZeneca.

LRRC8A anion channels modulate vascular reactivity via association with myosin phosphatase rho interacting protein.

Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 μM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.

RhoA/ROCK1 regulates the mitochondrial dysfunction through Drp1 induced by Porphyromonas gingivalis in endothelial cells.

Porphyromonas gingivalis (P. gingivalis) is a pivotal pathogen of periodontitis. Our previous studies have confirmed that mitochondrial dysfunction in the endothelial cells caused by P. gingivalis was dependent on Drp1, which may be the mechanism of P. gingivalis causing endothelial dysfunction. Nevertheless, the signalling pathway induced the mitochondrial dysfunction remains unclear. The purpose of this study was to investigate the role of the RhoA/ROCK1 pathway in regulating mitochondrial dysfunction caused by P. gingivalis. P. gingivalis was used to infect EA.hy926 cells (endothelial cells). The expression and activation of RhoA and ROCK1 were assessed by western blotting and pull-down assay. The morphology of mitochondria was observed by mitochondrial staining and transmission electron microscopy. Mitochondrial function was measured by ATP content, mitochondrial DNA and mitochondrial permeability transition pore openness. The phosphorylation and translocation of Drp1 were evaluated using western blotting and immunofluorescence. The role of the RhoA/ROCK1 pathway in mitochondrial dysfunction was investigated using RhoA and ROCK1 inhibitors. The activation of RhoA/ROCK1 pathway and mitochondrial dysfunction were observed in P. gingivalis-infected endothelial cells. Furthermore, RhoA or ROCK1 inhibitors partly prevented mitochondrial dysfunction caused by P. gingivalis. The increased phosphorylation and mitochondrial translocation of Drp1 induced by P. gingivalis were both blocked by RhoA and ROCK1 inhibitors. In conclusion, we demonstrate that the RhoA/ROCK1 pathway was involved in mitochondrial dysfunction caused by P. gingivalis by regulating the phosphorylation and mitochondrial translocation of Drp1. Our research illuminated a possible new mechanism by which P. gingivalis promotes endothelial dysfunction.

Rho-associated protein kinase 1 inhibition in hepatocytes attenuates nonalcoholic steatohepatitis.

NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis. Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis. Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH.

ROCK (RhoA/Rho Kinase) Activation in Atrial Fibrillation: Molecular Pathways and Clinical Implications.

Among the complex mechanisms of AF pathogenesis, intracellular calcium overload and oxidative stress play a major role, both triggered by inflammatory processes. The additional basic event taking place in AF is atrial fibrotic remodeling, again triggered by oxidative stress, which is determined by connexins rearrangement and differentiation of fibroblasts into active collagensecreting myofibroblasts. RhoA/ROCK system is the final pathway of a wide spectrum of molecular effectors such as Angiotensin II, platelet-derived growth factor, connective tissue growth factor and transforming growth factor β, that overall determine calcium dysregulation and pro-fibrotic remodeling. Both in experimental and clinical studies, RhoA/ROCK activation has been linked to superoxide ion production, fibrotic remodeling and connexins rearrangement, with important consequences for AF pathogenesis. ROCK pathway inhibition may therefore be a therapeutic or preventive target for special AF subgroups of patients.