MiRNA-6870-3p Regulates Lipopolysaccharide Induced Epicardial Adipose Tissue Inflammatory Genes via Targeting Tollip-Mediated JNK and NF-κB Signaling in Coronary Artery Disease.

MiR-6870-3p acts as a crucial regulator of gene expression at the posttranscriptional level and participates in immune responses. However, the roles of miR-6870-3p and its target genes and their underlying mechanisms in the inflammatory responses of epicardial adipose tissues (EATs) are unknown.MiRNA microarray was used to collect the miRNA expression profiles of EATs from five patients with coronary artery disease (CAD) and four individuals without CAD (n-CAD). Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to examine the expression of miR-6870-3p in the EATs. The mRNA and protein expression levels of Tollip and the key genes of the Toll-like receptor 4 (TLR4) signaling pathway were examined by qRT-PCR and Western blot analysis. The levels of inflammatory factors in the cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). We used a dual-luciferase reporter assay to validate the target gene of miR-6870-3p. The protein expression levels of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) were measured by Western blot analysis.Our results showed that miR-6870-3p was higher in the CAD EATs than in the n-CAD EATs. MiR-6870-3p was positively correlated with TLR4, interleukin (IL)-6, JNK, NF-κB (p65), and tumor necrosis factor (TNF)-α in the CAD EAT samples. Lipopolysaccharide (LPS) treatment upregulated the miR-6870-3p expression and downregulated the Tollip expression in the macrophages. When the macrophages were stimulated with LPS, MiR-6870-3p upregulation also aggravated the production of proinflammatory cytokines. The result of the luciferase reporter assays confirmed that miR-6870-3p directly targets Tollip. Moreover, miR-6870-3p upregulation in the macrophages resulted in the activation of the JNK/NF-κB pathway.Our study showed that miR-6870-3p regulates human EAT inflammation by targeting the Tollip-mediated JNK and NF-κB signaling pathways.

Potential osmoprotective roles of branchial heat shock proteins towards Na+, K+-ATPase in milkfish (Chanos chanos) exposed to hypotonic stress.

In euryhaline teleosts, osmoregulatory mechanisms vary with osmotic stresses, and heat shock proteins (HSPs) play a central role in maintaining cellular homeostasis. The present study aimed to investigate the expression and potential roles of HSP70 and HSP90 in the gills of seawater (SW)- and freshwater (FW)-acclimated milkfish (Chanos chanos). Four HSP genes, including Cchsc70 (heat shock cognate 70), Cchsp70, Cchsp90α, and Cchsp90ß, were analyzed in milkfish gills. Among these genes, only the mRNA abundance of branchial Cchsp90α was significantly lower in the FW-acclimated than in the SW-acclimated milkfish. Immunoblotting showed no significant difference in the relative protein abundance of branchial HSP70 and HSP90 between the two groups. The time-course experiments (from SW to FW) showed that the protein abundance of HSP70 and HSP90 at the 3 h and 6 h post-transfer and then declined gradually. To further illustrate the potential osmoregulatory roles of HSP70 and HSP90, their interaction with Na+, K+-ATPase (NKA, the primary driving force for osmoregulation) was analyzed using co-immunoprecipitation. The results showed the interaction between HSP70, HSP90 and NKA after acclimation to SW or FW increased within 3 h; and then returned to normal levels within 7 days. To our knowledge, the present study was the first to demonstrate that the interaction between HSP70, HSP90 and NKA changes with hypotonic stress in euryhaline teleosts. Before the transfer, no interaction was detected. When transferred to FW from SW, the interaction of HSP70 and HSP90 with NKA were detected. The results suggested that HSP70 and HSP90 participated in the acute responses of osmoregulatory mechanisms to protect branchial NKA from hypotonic stress in milkfish.

Positive correlation of gene expression between branchial FXYD proteins and Na+/K+-ATPase of euryhaline milkfish in response to hypoosmotic challenges.

FXYD proteins are crucial regulators of Na+/K+-ATPase (NKA), which plays an important role in ion exchange by providing the driving force for other ion-transporting systems in the osmoregulatory organs, including the gills. In milkfish (Chanos chanos), gill NKA has been widely investigated and found to alter its expression (both mRNA and protein) and activity in response to environmental salinity changes. However, the expression and roles of the regulatory proteins of NKA, the FXYD proteins, in milkfish gills upon salinity challenge is not yet clear. Hence, this study illustrated the potential roles of milkfish branchial FXYD proteins in modulating NKA expression via identification and tissue distributions of FXYD proteins, as well as the effects of salinity on expression of gill fxyd and nka mRNA. Six milkfish FXYD proteins (CcFXYD) were identified. In milkfish gill, gill-specific Ccfxyd11 was the predominant member, followed by Ccfxyd9 and Ccfxyd8. Upon hypoosmotic challenges, increases in gill Ccfxyd11, Ccfxyd8, Ccnka α1, and Ccnka ß1 mRNA as well as significantly positive correlations were observed. Moreover, after acute salinity changes, expression of gill Ccfxyd11 and Ccnka was found to change with ambient salinity, and significant positive correlations were also exhibited between Ccfxyd11 and Ccnka α1. Overall, these results revealed close relationships between CcFXYD11 and CcNKA α1 in milkfish gills, highlighting the potential roles of CcFXYD11 in osmoregulation.

A Stenohaline Medaka, Oryzias woworae, Increases Expression of Gill Na(+), K(+)-ATPase and Na(+), K(+), 2Cl(-) Cotransporter 1 to Tolerate Osmotic Stress.

The present study aimed to evaluate the osmoregulatory mechanism of Daisy's medaka, O. woworae,as well as demonstrate the major factors affecting the hypo-osmoregulatory characteristics of euryhaline and stenohaline medaka. The medaka phylogenetic tree indicates that Daisy's medaka belongs to the celebensis species group. The salinity tolerance of Daisy's medaka was assessed. Our findings revealed that 20‰ (hypertonic) saltwater (SW) was lethal to Daisy's medaka. However, 62.5% of individuals survived 10‰ (isotonic) SW with pre-acclimation to 5‰ SW for one week. This transfer regime, "Experimental (Exp.) 10‰ SW", was used in the following experiments. After 10‰ SW-transfer, the plasma osmolality of Daisy's medaka significantly increased. The protein abundance and distribution of branchial Na(+), K(+)-ATPase (NKA) and Na(+), K(+), 2Cl(-) cotransporter 1 (NKCC1) were also examined after transfer to 10‰ SW for one week. Gill NKA activity increased significantly after transfer to 10‰ SW. Meanwhile, elevation of gill NKA αα-subunit protein-abundance was found in the 10‰ SW-acclimated fish. In gill cross-sections, more and larger NKA-immunoreactive (NKA-IR) cells were observed in the Exp. 10‰ SW medaka. The relative abundance of branchial NKCC1 protein increased significantly after transfer to 10‰ SW. NKCC1 was distributed in the basolateral membrane of NKA-IR cells of the Exp. 10‰ SW group. Furthermore, a higher abundance of NKCC1 protein was found in the gill homogenates of the euryhaline medaka, O. dancena, than in that of the stenohaline medaka, O. woworae.

FXYD11 mediated modulation of Na(+)/K(+)-ATPase activity in gills of the brackish medaka (Oryzias dancena) when transferred to hypoosmotic or hyperosmotic environments.

FXYD proteins regulate Na(+)/K(+)-ATPase (NKA), which is a primary active pump that provides the driving force that triggers osmoregulatory systems in teleosts. To explore the regulatory mechanisms between FXYD and NKA in euryhaline teleosts, the expression of NKA (mRNA, protein, and activity) and FXYD11 and their interaction were examined in the gills of brackish medaka (Oryzias dancena) when transferred from brackish water (BW; 15‰) to fresh water (FW) or seawater (SW; 35‰). The mRNA expression of Odfxyd11 and Odnka-α was elevated 48h post-hypoosmotic transfer. Moreover, FXYD11 protein and NKA activity were upregulated 12h after transfer to FW. When transferred to SW, the protein abundance of FXYD11 and the NKA α-subunit did not show apparent changes, while Odfxyd11 and Odnka-α mRNA expression and NKA activity increased significantly 12h and 1h post-transfer, respectively. To clarify the FXYD11 mechanisms involved in modulating NKA activity via their interaction, co-immunoprecipitation was further applied to O. dancena gills. The results revealed that the levels of protein-protein interaction between branchial NKA and FXYD11 increased acutely 12h after the transfer from BW to FW. However, immediate upregulation of NKA activity 1h following post-exposure to SW, without the elevation of protein-protein interaction levels, was found. Hence, branchial NKA activity of O. dancena was suggested to be rapidly regulated by FXYD11 interaction with NKA when acclimated to hypoosmotic environments. To the best of our knowledge, this is the first study that focuses on the efficacy of interactions between FXYD11 and NKA in the gills of euryhaline teleosts.

Impaired Na(+)/K(+)-ATPase Function in Patients with Interstitial Cystitis/Painful Bladder Syndrome.

Na(+)/K(+)-ATPase (NKA) is abundantly expressed in the basolateral membrane of epithelial cells, which is necessary for tight junction formation. The tight junction is an urothelial barrier between urine and the underlying bladder. Impairment of tight junctions allows migration of urinary solutes in patients with interstitial cystitis/painful bladder syndrome (IC/PBS). We evaluated NKA expression and activity in bladder samples from patients with IC/PBS. The study group consisted of 85 patients with IC/PBS, and the control group consisted of 20 volunteers. Bladder biopsies were taken from both groups. We determined the expression and distribution of NKA using NKA activity assays, immunoblotting, immunohistochemical staining, and immunofluorescent staining. The protein levels and activity of NKA in the study group were significantly lower than the control group (1.08 ± 0.06 vs. 2.39 ± 0.29 and 0.60 ± 0.04 vs. 1.81 ± 0.18 µmol ADP/mg protein/hour, respectively; P < 0.05). Additionally, immunofluorescent staining for detection of CK7, a marker of the bladder urothelium, predominantly colocalized with NKA in patients in the study group. Our results demonstrated the expression and activity of NKA were decreased in bladder biopsies of patients with IC/PBS. These findings suggest that NKA function is impaired in the bladders from patients with IC/PBS.

Secretion of adipocytes and macrophages under conditions of inflammation and/or insulin resistance and effect of adipocytes on preadipocytes under these conditions.

The purpose of the present study was to examine changes in preadipocytes following the coculture of preadipocytes and adipocytes and the effects on the secretion of adipocytes and macrophages following induction of inflammation and insulin resistance. Mature adipocytes and RAW264.7 macrophages were treated with lipopolysaccharide and insulin to establish models of inflammation and insulin resistance, respectively. The mRNA expression levels of IL-6, MCP-1, and TNF-α in all adipocyte treatment groups were significantly greater compared with the control, and that of adiponectin was less (P<0.05). In the RAW264.7 macrophages, the mRNA expression levels of IL-6 and TNF-α were greater than those in the control group (P<0.05). Moreover, the results of this study confirmed that adipocytes and macrophages increased the secretion of inflammatory factors under conditions of induced inflammation and insulin resistance. In addition, 3T3-L1 adipocytes inhibited the proliferation and differentiation of preadipocytes when cocultured with adipocytes under conditions of inflammation and/or insulin resistance, and the phenotype of preadipocytes did not change.

Exploration of the mechanisms of protein quality control and osmoregulation in gills of Chromis viridis in response to reduced salinity.

Fish gills are the vital multifunctional organ in direct contact with external environment. Therefore, activation of the cytoprotective mechanisms to maintain branchial cell viability is important for fish upon stresses. Salinity is one of the major factors strongly affecting cellular and organismal functions. Reduction of ambient salinity may occur in coral reef and leads to osmotic stress for reef-associated stenohaline fish. However, the physiological responses to salinity stress in reef-associated fish were not examined substantially. With this regard, the physiological parameters and the responses of protein quality control (PQC) and osmoregulatory mechanisms in gills of seawater (SW; 33-35 ‰)- and brackish water (BW; 20 ‰)-acclimated blue-green damselfish (Chromis viridis) were explored. The results showed that the examined physiological parameters were maintained within certain physiological ranges in C. viridis acclimated to different salinities. In PQC mechanism, expression of heat-shock protein (HSP) 90, 70, and 60 elevated in response to BW acclimation while the levels of ubiquitin-conjugated proteins were similar between the two groups. Thus, it was presumed that upregulation of HSPs was sufficient to prevent the accumulation of aggregated proteins for maintaining the protein quality and viability of gill cells when C. viridis were acclimated to BW. Moreover, gill Na(+)/K(+)-ATPase expression and protein amounts of basolaterally located Na(+)/K(+)/2Cl(-) cotransporter were higher in SW fish than in BW fish. Taken together, this study showed that the cytoprotective and osmoregulatory mechanisms of blue-green damselfish were functionally activated and modulated to withstand the challenge of reduction in salinity for maintaining physiological homeostasis.

The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments.

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl(-) through the transporters including the Na(+), K(+)-ATPase (NKA), Na(+), K(+), 2Cl(-) cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl(-) secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (>1day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24h, the Cl(-) secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.

Salinity-dependent changes in branchial morphometry and Na+ , K+ -ATPase responses of euryhaline Asian sea bass, Lates calcarifer.

Asian sea bass (Lates calcarifer Bloch, 1790) is a euryhaline fish widely cultured in Asia and Australia. Although it is common to culture Asian sea bass at different salinities, osmoregulatory responses of Asian sea bass during acclimation to various salinities have not been fully observed. In this study, we used scanning electron microscopy to observe the morphology of the ionocyte apical membrane of Asian sea bass acclimated to fresh water (FW), 10‰ brackish water (BW10), 20‰ brackish water (BW20), and seawater (SW; 35‰). Three types of ionocytes were identified in FW and BW fish: (I) flat type with microvilli, (II) basin type with microvilli, and (III) small- hole type. Flat type I ionocytes were also observed in the lamellae of the FW fish. In contrast, two types of ionocytes were identified in SW fish: (III) small-hole type and (IV) big-hole type. Furthermore, we observed Na+ , K+ -ATPase (NKA) immunoreactive cells in the gills, which represent the localization of ionocytes. The highest protein abundance was observed in the SW and FW groups, whereas the highest activity was observed in the SW group. In contrast, the BW10 group had the lowest protein abundance and activity. This study demonstrates the effects of osmoregulatory responses on the morphology and density of ionocytes, as well as protein abundance and activity of NKA. In this study, we found that Asian sea bass had the lowest osmoregulatory response in BW10, because the lowest amounts of ionocytes and NKA were required to maintain osmolality at this salinity.

Increased expression of hypoxia-inducible factor-1alpha and Bcl-2 in varicocele and varicose veins.

BACKGROUND: Primary vein wall abnormalities leading to secondary blood stasis and increased venous pressure that cause tissue hypoxia are observed in varicocele and varicose veins. Both types of diseased vessels are characterized by dilated thickened vein walls. Hypoxia upregulates Bcl-2 (antiapoptosis protein) expression in different human cell types. We studied the expression of hypoxia-inducible factor-1alpha (HIF-1α) and Bcl-2 in both venous diseases. METHODS: All vascular specimens, including the saphenous and internal spermatic veins, from patients with either varicocele or left inguinal herniorrhaphy (control group) were studied using immunoblotting, immunohistochemical staining, and double immunofluorescence staining. The data were analyzed using 1-way analysis of variance with Tukey comparison test. RESULTS: Protein analysis revealed that both venous diseases had a higher expression of HIF-1α and Bcl-2 compared with the control group (P < 0.05). Immunohistochemical staining and double immunofluorescence staining revealed that the greatest degree of HIF-1α and Bcl-2 colocalization occurred in the muscle layer of both diseased vessels. Moreover, under confocal microscopy, elevated Bcl-2 expression was found in the endothelium of both study groups compared with the control group. CONCLUSIONS: Our findings revealed increased expression of HIF-1α and Bcl-2 in varicocele and varicose veins and increased Bcl-2 expression especially in the endothelium under hypoxia. Thus, Bcl-2 overexpression may protect cells against apoptosis and contribute to the dilated thickened walls seen in both types of diseased vessels.

Quietness of circular RNA circ_0054633 alleviates the inflammation and proliferation in lipopolysaccharides-induced acute lung injury model through NF-κB signaling pathway.

AIM: Acute lung injury (ALI) is the mild form of acute respiratory distress syndrome (ARDS) which is a common lung disease with a high incidence and mortality rate. Recent studies manifested that some circular RNAs were associated with ALI. In this study, we aimed to uncover the effect of circular RNA circ_0054633 on ALI initiation and progression and proposed a new mechanism related to ALI. METHODS: The lipopolysaccharides (LPS)-induced acute lung injury model were build both in vivo of rat and in vitro of primary murine pulmonary microvascular endothelial cells (MPVECs). Hematoxylin and eosin (H&E) was employed to observe the tissue morphology and estimate the degree of lung damage. We used real-time quantitative polymerase chain reaction (RT-qPCR) to measure the expression level of circ_0054633. The expression levels of inflammatory cytokines IL-17A and tumor necrosis factor-α (TNF-α) were detected by ELISA. The effects of circ_0054633 on MPVECs proliferation and apoptosis were detected with the help of CCK-8 and apoptosis assay, separately. The expression level of NF-κB p65 protein was measured by Western blot. RESULTS: circ_0054633, IL-17A, TNF-α and NF-κB p65 were all overexpressed in LPS-treated rat and MPVECs, and LPS enhanced the proliferation and apoptosis of MPVECs. While circ_0054633 silencing reversed the above promotion effects of LPS on IL-17A, TNF-α expression and MPVECs proliferation and apoptosis. CONCLUSIONS: Quietness of circ_0054633 alleviated LPS-induced ALI via NF-κB signaling pathway, implicating circ_0054633 may be a potential biomarker for diagnose and therapy of ALI.

Involved intrinsic apoptotic pathway in the varicocele and varicose veins.

BACKGROUND: Disordered programmed cell death may play a role in the development of venous diseases. Tissue hypoxia caused by blood stagnation and venous hypertension is the similar etiology of varicocele and varicose veins. We studied the vascular histopathology and determined whether there is the same apoptotic pathway in both venous diseases. METHODS: The study groups consisted of 1-cm venous segments obtained from 10 patients during vascular stripping surgery for varicose saphenous vein and 1 cm of internal spermatic veins obtained from 12 patients during left varicocele repair. The control samples of 1 cm internal spermatic vein were obtained from 10 male patients who underwent left inguinal herniorrhaphy. The three layers of vascular histology were measured and compared by Masson trichrome stain, and the apoptotic proteins including Bcl-2, Fas, cleaved caspase-9, cleaved caspase-8, and cleaved caspase-3 were detected. Data were analyzed using the one-way analysis of variance with Tukey's comparison test. RESULTS: The relative thickness of intima and adventitia layer was smaller in both study groups than in the control group. But a significant hypertrophy of media layer was observed in the varicocele and varicose veins than in the control group (p < 0.05). Overexpression of Bcl-2 and decreased expressions of cleaved caspase-9 and cleaved caspase-3 was observed in both study groups. There is no statistical difference in Fas and cleaved caspase-8 expressions in the control and study groups. CONCLUSION: Our data showed vascular smooth muscle hypertrophy in the diseased vessels. The same dysregulation of apoptosis through intrinsic pathway was demonstrated in varicocele and varicose veins under tissues hypoxia. This mechanism of reduced apoptosis might contribute to the dilated and thickened walls of both venous diseases.

Environmentally relevant concentrations of di(2-ethylhexyl)phthalate exposure alter larval growth and locomotion in medaka fish via multiple pathways.

Di(2-ethylhexyl)phthalate (DEHP) is a commonly used plasticizer, with evidence of ubiquitous human exposure and widespread occurrence in the aquatic environment. It is an emerging environmental pollutant with regulatory priority; however, most studies have focused on the toxicity of DEHP related to endocrine disruption and reproduction in mammals. The ecotoxicological impact of phthalates (e.g., DEHP) on early life stages of fish under environmentally relevant concentrations of chronic exposure remains unclear. In this study, 7-day post-hatching fry of medaka fish (Oryzias latipes) underwent 21-day continuous exposure to DEHP solutions at 20, 100 and 200 µg/L to assess the effects on fish development and locomotion and related toxic mechanisms. Larval mortality was low with DEHP (20-200 µg/L) within 21 days, but such exposure significantly reduced fish body weight and length and altered swimming behavior. At 21 days, DEHP exposure resulted in specific patterns of larval locomotion (e.g., increased maximum velocity and absolute turn angle) and dose-dependently increased the mRNA expression of acetylcholinesterase (ache) but did not alter AChE activity. Transcriptional expression of antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase and peroxisome proliferation-activated receptor and retinoid X receptor genes was significantly suppressed with 21-day DEHP exposure (20-200 µg/L), with marginal alteration in reactive oxygen species levels and antioxidant activities within the dosing period. As well, DEHP altered the mRNA expression of p53-regulated apoptosis pathways, such as upregulated p53, p21 and bcl-2 and downregulated caspase-3 expression, with increased enzymatic activity of caspase-3 in larvae. Our results suggest that toxic mechanisms of waterborne DEHP altered fish growth and locomotion likely via a combined effect of oxidative stress, neurotoxicity and apoptosis pathways.

Corrigendum: MicroRNA-200a/200b Modulate High Glucose-Induced Endothelial Inflammation by Targeting O-linked N-Acetylglucosamine Transferase Expression.

[This corrects the article on p. 355 in vol. 9, PMID: 29720943.].

Illicit drug ketamine induces adverse effects from behavioral alterations and oxidative stress to p53-regulated apoptosis in medaka fish under environmentally relevant exposures.

With increasing problems of drug abuse worldwide, aquatic ecosystems are contaminated by human pharmaceuticals from the discharge of hospital or municipal effluent. However, ecotoxicity data and related toxic mechanism for neuroactive controlled or illicit drugs are still lacking, so assessing the associated hazardous risk is difficult. This study aims to investigate the behavioral changes, oxidative stress, gene expression and neurotoxic or apoptosis effect(s) in larvae of medaka fish (Oryzias latipes) with environmentally relevant exposures of ketamine (KET) solutions for 1-14 days. KET exposure at an environmentally relevant concentration (0.004 µM) to 40 µM conferred specific patterns in larval swimming behavior during 24 h. At 14 days, such exposure induced dose- and/or time-dependent alteration on reactive oxygen species induction, the activity of antioxidants catalase and superoxide dismutase, glutathione S-transferase and malondialdehyde contents in fish bodies. KET-induced oxidative stress disrupted the expression of acetylcholinesterase and p53-regulated apoptosis pathways and increased caspase expression in medaka larvae. The toxic responses of medaka larvae, in terms of chemical effects, were qualitatively analogous to those of zebrafish and mammals. Our results implicate a toxicological impact of waterborne KET on fish development and human health, for potential ecological risks of directly releasing neuroactive drugs-containing wastewater into the aquatic environment.

MicroRNA-200a/200b Modulate High Glucose-Induced Endothelial Inflammation by Targeting O-linked N-Acetylglucosamine Transferase Expression.

Background and Aims: Increased O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins by O-GlcNAc transferase (OGT) is associated with diabetic complications. Furthermore, oxidative stress promotes endothelial inflammation during diabetes. A previous study reported that microRNA-200 (miR-200) family members are sensitive to oxidative stress. In this study, we examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation. Methods: HAECs were stimulated with high glucose (25 mM) for 12 and 24 h. Real-time polymerase chain reaction (PCR), western blotting, THP-1 adhesion assay, bioinformatics predication, transfection of miR-200a/200b mimic or inhibitor, luciferase reporter assay, and transfection of siRNA OGT were performed. The aortic endothelium of db/db diabetic mice was evaluated by immunohistochemistry staining. Results: HG upregulated OGT mRNA and protein expression and protein O-GlcNAcylation levels (RL2 antibody) in HAECs, and showed increased intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Bioinformatics analysis revealed homologous sequences between members of the miR-200 family and the 3'-untranslated region (3'-UTR) of OGT mRNA, and real-time PCR analysis confirmed that members of miR-200 family were significantly decreased in HG-stimulated HAECs. This suggests the presence of an impaired feedback restraint on HG-induced endothelial protein O-GlcNAcylation levels because of OGT upregulation. A luciferase reporter assay demonstrated that miR-200a/200b mimics bind to the 3'-UTR of OGT mRNA. Transfection with miR-200a/200b mimics significantly inhibited HG-induced OGT mRNA expression, OGT protein expression; protein O-GlcNAcylation levels; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Additionally, siRNA-mediated OGT depletion reduced HG-induced protein O-GlcNAcylation; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion, confirming that HG-induced endothelial inflammation is partially mediated via OGT-induced protein O-GlcNAcylation. These results were validated in vivo: tail-vein injection of miR-200a/200b mimics downregulated endothelial OGT and ICAM-1 expression in db/db mice. Conclusion: miR-200a/200b are involved in modulating HG-induced endothelial inflammation by regulating OGT-mediated protein O-GlcNAcylation, suggesting the therapeutic role of miR-200a/200b on vascular complications in diabetes.

Intestinal FXYD12 and sodium-potassium ATPase: A comparative study on two euryhaline medakas in response to salinity changes.

FXYD proteins are the regulators of sodium-potassium ATPase (Na+/K+-ATPase, NKA). In teleosts, NKA is a primary driving force for the operation of many ion transport systems in the osmoregulatory organs (e.g. intestines). Hence, the purpose of this study was to determine the expression of FXYD proteins and NKA α-subunit in the intestines of two closely related medakas (Oryzias dancena and O. latipes), which came from different salinity habitats and have diverse osmoregulatory capabilities, to illustrate the association between NKA and FXYD proteins of two medaka species in response to salinity changes. The results showed that the fxyd12 mRNA was the most predominant in the intestines of both medakas. The association of FXYD12 and NKA in the intestines of the two medaka species was demonstrated via double immunofluorescent staining and co-immunoprecipitation. Upon salinity challenge, the localization of FXYD12 and NKA was similar in the intestines of the two medaka species. However, the expression profiles of intestinal FXYD12 and NKA (mRNA and protein levels), as well as NKA activity differed between the medakas. These results showed that FXYD12 may play a role in modulating NKA activity in the intestines of the two medakas following salinity changes in the maintenance of internal homeostasis. These findings contributed to knowledge of the expression and potential role of vertebrate FXYD12, the regulators of NKA, upon salinity challenge.

Increased expression of the sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins.

Objectives Varicocele is characterized by dilatation and tortuosity of the internal spermatic vein. Sonic hedgehog plays an important role in angiogenesis and vascular remodeling under hypoxic stress. We studied the relationship and distribution of SHH and vascular endothelial growth factor in internal spermatic vein in patients diagnosed with varicocele. Methods Specimens of 1 cm were taken from the internal spermatic vein during left varicocele repair (N = 20). The control samples of ISV were obtained from eight male patients who underwent left inguinal herniorrhaphy. We analyzed the sonic hedgehog and vascular endothelial growth factor expression and distribution by immunoblotting, immunohistochemistry, immunofluorescent staining, and confocal laser scanning microscopy. The data were analyzed using the Student's t test. Results Immunoblotting showed higher expression of sonic hedgehog and vascular endothelial growth factor proteins in varicocele veins than in the control group ( P < 0.05) which located over muscle layer and endothelium was demonstrated by immunohistochemical staining. Both proteins with co-localization in the muscle layer and especially distributed in endothelium of varicocele veins were revealed under confocal microscopy. Conclusions These findings showed the upexpression of sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins which imply that the reducing hypoxia or using sonic hedgehog antagonists may be helpful for this vascular disease.

Na+, K+-ATPase ß1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na+, K+-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and ß-subunits. Among the NKA genes, α1-ß1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-ß1 isozyme. Based on the sequenced milkfish transcriptome, an NKA ß1 subunit gene was identified that had the highest amino acid homology to ß233, a NKA ß1 subunit paralog originally identified in the eel. Despite this high level of homology to ß233, phylogenetic analysis and the fact that only a single NKA ß1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA ß1 subunit gene. The results of accurate domain prediction of the ß1 subunit, co-localization of α1 and ß1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and ß1 subunits, indicated the formation of a α1-ß1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and ß1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA ß1 subunit and provided the first evidence for an NKA α1-ß1 association in gill ionocytes of euryhaline teleosts.