Ezrin Is a Novel Protein Partner of Aquaporin-5 in Human Salivary Glands and Shows Altered Expression and Cellular Localization in Sjögren’s Syndrome

Sjögren’s syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5–ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5–ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5–ezrin co-localization. Our data reveal that AQP5–ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients     

Nitro-Oleic Acid (NO2-OA) Improves Systolic Function in Dilated Cardiomyopathy by Attenuating Myocardial Fibrosis

Nitro-oleic acid (NO₂-OA), a nitric oxide (NO)- and nitrite (NO₂⁻)-derived electrophilic fatty acid metabolite, displays anti-inflammatory and anti-fibrotic signaling actions and therapeutic benefit in murine models of ischemia-reperfusion, atrial fibrillation, and pulmonary hypertension. Muscle LIM protein-deficient mice (Mlp^−/−) develop dilated cardiomyopathy (DCM), characterized by impaired left ventricular function and increased ventricular fibrosis at the age of 8 weeks. This study investigated the effects of NO₂-OA on cardiac function in Mlp^−/− mice both in vivo and in vitro. Mlp^−/− mice were treated with NO₂-OA or vehicle for 4 weeks via subcutaneous osmotic minipumps. Wildtype (WT) littermates treated with vehicle served as controls. Mlp^−/− mice exhibited enhanced TGFβ signalling, fibrosis and severely reduced left ventricular systolic function. NO₂-OA treatment attenuated interstitial myocardial fibrosis and substantially improved left ventricular systolic function in Mlp^−/− mice. In vitro studies of TGFβ-stimulated primary cardiac fibroblasts further revealed that the anti-fibrotic effects of NO₂-OA rely on its capability to attenuate fibroblast to myofibroblast transdifferentiation by inhibiting phosphorylation of TGFβ downstream targets. In conclusion, we demonstrate a substantial therapeutic benefit of NO₂-OA in a murine model of DCM, mediated by interfering with endogenously activated TGFβ signaling.     

Resveratrol Treatment Prevents Increase of Mast Cells in Both Murine OVA Enteritis and IL-10−/− Colitis

Mast cells are involved in allergic and other inflammatory diseases. The polyphenol resveratrol is known for its anti-inflammatory properties and may be used as nutraceutical in mast cell associated diseases. We analyzed the effect of resveratrol on mast cells in vivo in ovalbumin-induced allergic enteritis as well as experimental colitis in IL-10^−/− mice which received resveratrol via drinking water. Treatment with resveratrol prevented the increase in mast cells in both allergic enteritis and chronic colitis in duodenum as well as in colon. Further, it delayed the onset of diseases symptoms and ameliorated diseases associated parameters such as tissue damage as well as inflammatory cell infiltration in affected colon sections. In addition to the findings in vivo, resveratrol inhibited IgE-dependent degranulation and expression of pro-inflammatory cytokines such as TNF-α in IgE/DNP-activated as well as in LPS-activated bone marrow-derived mast cells. These results indicate that resveratrol may be considered as an anti-allergic and anti-inflammatory plant-derived component for the prevention or treatment of mast cell-associated disorders of the gastrointestinal tract.     

Short-Term Pharmacological Induction of Arterial Stiffness and Hypertension with Angiotensin II Does Not Affect Learning and Memory and Cerebral Amyloid Load in Two Murine Models of Alzheimer’s Disease

Given the unprecedented rise in the world’s population, the prevalence of prominent age-related disorders, like cardiovascular disease and dementia, will further increase. Recent experimental and epidemiological evidence suggests a mechanistic overlap between cardiovascular disease and dementia with a specific focus on the linkage between arterial stiffness, a strong independent predictor of cardiovascular disease, and/or hypertension with Alzheimer’s disease. In the present study, we investigated whether pharmacological induction of arterial stiffness and hypertension with angiotensin II (1 µg·kg⁻¹·min⁻¹ for 28 days via an osmotic minipump) impairs the progression of Alzheimer’s disease in two mouse models (hAPP23^+/− and hAPPswe/PSEN1dE9 mice). Our results show increased arterial stiffness in vivo and hypertension in addition to cardiac hypertrophy after angiotensin II treatment. However, visuospatial learning and memory and pathological cerebral amyloid load in both Alzheimer’s disease mouse models were not further impaired. It is likely that the 28-day treatment period with angiotensin II was too short to observe additional effects on cognition and cerebral pathology.     

Mechanism of GAPDH Redox Signaling by H2O2 Activation of a Two−Cysteine Switch

Oxidation of glyceraldehyde−3−phosphate dehydrogenase (GAPDH) by reactive oxygen species such as H₂O₂ activate pleiotropic signaling pathways is associated with pathophysiological cell fate decisions. Oxidized GAPDH binds chaperone proteins with translocation of the complex to the nucleus and mitochondria initiating autophagy and cellular apoptosis. In this study, we establish the mechanism by which H₂O₂−oxidized GAPDH subunits undergo a subunit conformational rearrangement. H₂O₂ oxidizes both the catalytic cysteine and a vicinal cysteine (four residues downstream) to their respective sulfenic acids. A ‘two−cysteine switch’ is activated, whereby the sulfenic acids irreversibly condense to an intrachain thiosulfinic ester resulting in a major metastable subunit conformational rearrangement. All four subunits of the homotetramer are uniformly and independently oxidized by H₂O₂, and the oxidized homotetramer is stabilized at low temperatures. Over time, subunits unfold forming disulfide−linked aggregates with the catalytic cysteine oxidized to a sulfinic acid, resulting from thiosulfinic ester hydrolysis via the highly reactive thiosulfonic ester intermediate. Molecular Dynamic Simulations provide additional mechanistic insights linking GAPDH subunit oxidation with generating a putative signaling conformer. The low−temperature stability of the H₂O₂−oxidized subunit conformer provides an operable framework to study mechanisms associated with gain−of−function activities of oxidized GAPDH to identify novel targets for the treatment of neurodegenerative diseases.     

Unique Regulation of Intestinal Villus Epithelial Cl−/HCO3− Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl⁻/HCO₃⁻ exchange, measured as DIDS-sensitive ³⁶Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered K_m with no effects on V_max. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl⁻/HCO₃⁻ exchangers, were unaltered. Thus, inhibition of villus cell Cl⁻/HCO₃⁻ exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl⁻, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.     

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.     

Simultaneous Monitoring of pH and Chloride (Cl−) in Brain Slices of Transgenic Mice

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl⁻]_i) and hydrogen ([H⁺] _i) the construct, called ClopHensor, which consists of a H⁺- and Cl⁻-sensitive variant of the enhanced green fluorescent protein (E²GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H⁺]_i and [Cl⁻]_i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H⁺]_i and [Cl⁻]_i under different experimental conditions including changing of external concentrations of ions (Ca²⁺, Cl⁻, K⁺, Na⁺) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl⁻ and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl⁻ and pH.     

Fucose Ameliorates Tritrichomonas sp.-Associated Illness in Antibiotic-Treated Muc2−/− Mice

The mucus layer in the intestine plays a critical role in regulation of host–microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2^−/− mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2^−/− mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host–microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value.     

Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

Alzheimer’s disease (AD) is associated with the accumulation and aggregation of amyloid in the brain. The cation channel TRPV2 may mediate the pathological changes in mild cognitive impairment. A high-affinity agonist of TRPV2 named cannabidiol is one of the candidate drugs for AD. However, the molecular mechanism of cannabidiol via TRPV2 in AD remains unknown. The present study investigated whether cannabidiol enhances the phagocytosis and clearance of microglial Aβ via the TRPV2 channel. We used a human dataset, mouse primary neuron and microglia cultures, and AD model mice to evaluate TRPV2 expression and the ability of microglial amyloid-β phagocytosis in vivo and in vitro. The results revealed that TRPV2 expression was reduced in the cortex and hippocampus of AD model mice and AD patients. Cannabidiol enhanced microglial amyloid-β phagocytosis through TRPV2 activation, which increased the mRNA expression of the phagocytosis-related receptors, but knockdown of TRPV2 or Trem2 rescued the expression. TRPV2-mediated effects were also dependent on PDK1/Akt signaling, a pathway in which autophagy was indispensable. Furthermore, cannabidiol treatment successfully attenuated neuroinflammation while simultaneously improving mitochondrial function and ATP production via TRPV2 activation. Therefore, TRPV2 is proposed as a potential therapeutic target in AD, while CBD is a promising drug candidate for AD.     

Intracerebroventricular Treatment with 2-Hydroxypropyl-β-Cyclodextrin Decreased Cerebellar and Hepatic Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Expression in Niemann–Pick Disease Type C Model Mice

Niemann–Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-β-CD in Npc1 gene-deficient (Npc1^−/−) mice. Intracerebroventricular HP-β-CD inhibited cerebellar Purkinje cell damage in Npc1^−/− mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in Npc1^−/− mice. Repeated doses of intracerebroventricular HP-β-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of Npc1^−/− mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-β-CD treatment.     

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca²⁺ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2^−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.     

Embryonic-Derived Myb− Macrophages Enhance Bacterial Clearance and Improve Survival in Rat Sepsis

Peritoneal resident macrophages play a key role in combating sepsis in the peritoneal cavity. We sought to determine if peritoneal transplantation of embryonic Myb⁻ “peritoneal-like” macrophages attenuate abdominal fecal sepsis. Directed differentiation of rodent pluripotent stem cells (PSCs) was used in factor-defined media to produce embryonic-derived large “peritoneal-like” macrophages (Ed-LPM) that expressed peritoneal macrophage markers and demonstrated phagocytic capacity. Preclinical in vivo studies determined Ed-LPM efficacy in rodent abdominal fecal sepsis with or without Meropenem. Ex vivo studies explored the mechanism and effects of Ed-LPM on host immune cell number and function, including phagocytosis, reactive oxygen species (ROS) production, efferocytosis and apoptosis. Ed-LPM reduced sepsis severity by decreasing bacterial load in the liver, spleen and lungs. Ed-LPM therapy significantly improved animal survival by ~30% and reduced systemic bacterial burden to levels comparable to Meropenem therapy. Ed-LPM therapy decreased peritoneal TNFα while increasing IL-10 concentrations. Ed-LPMs enhanced peritoneal macrophage phagocytosis of bacteria, increased macrophage production of ROS and restored homeostasis via apoptosis and efferocytosis-induced clearance of neutrophils. In conclusion, Ed-LPM reduced systemic sepsis severity, improved survival and reduced bacterial load by enhancing peritoneal macrophage bacterial phagocytosis and killing and clearance of intra-peritoneal neutrophils. Macrophage therapy may be a potential strategy to address sepsis.     

Antimicrobial Polymer−Based Assemblies: A Review

An antimicrobial supramolecular assembly (ASA) is conspicuous in biomedical applications. Among the alternatives to overcome microbial resistance to antibiotics and drugs, ASAs, including antimicrobial peptides (AMPs) and polymers (APs), provide formulations with optimal antimicrobial activity and acceptable toxicity. AMPs and APs have been delivered by a variety of carriers such as nanoparticles, coatings, multilayers, hydrogels, liposomes, nanodisks, lyotropic lipid phases, nanostructured lipid carriers, etc. They have similar mechanisms of action involving adsorption to the cell wall, penetration across the cell membrane, and microbe lysis. APs, however, offer the advantage of cheap synthetic procedures, chemical stability, and improved adsorption (due to multipoint attachment to microbes), as compared to the expensive synthetic routes, poor yield, and subpar in vivo stability seen in AMPs. We review recent advances in polymer−based antimicrobial assemblies involving AMPs and APs.     

Male-Dependent Promotion of Colitis in 129 Rag2−/− Mice Co-Infected with Helicobacter pylori and Helicobacter hepaticus

The prevalence of gastric Helicobacter pylori (Hp) infection is ~50% of the world population. However, how Hp infection influences inflammatory bowel disease in humans is not fully defined. In this study, we examined whether co-infection with Hp influenced Helicobacter hepaticus (Hh)–induced intestinal pathology in Rag2^−/− mice. Rag2^−/− mice of both sexes were infected with Hh, of which a subgroup was followed by infection with Hp two weeks later. Co-infected males, but not females, had significantly higher total colitis index scores in the colon at both 10 and 21 weeks post-Hh infection (WPI) and developed more severe dysplasia at 21 WPI compared with mono-Hh males. There were no significant differences in colonization levels of gastric Hp and colonic Hh between sexes or time-points. In addition, mRNA levels of colonic Il-1β, Ifnγ, Tnfα, Il-17A, Il-17F, Il-18, and Il-23, which play important roles in the development and function of proinflammatory innate lymphoid cell groups 1 and 3, were significantly up-regulated in the dually infected males compared with mono-Hh males at 21 WPI. These data suggest that concomitant Hp infection enhances the inflammatory responses in the colon of-Hh-infected Rag2^−/− males, which results in more severe colitis and dysplasia.     

New Advances in Urea Transporter UT-A1 Membrane Trafficking

The vasopressin-regulated urea transporter UT-A1, expressed in kidney inner medullary collecting duct (IMCD) epithelial cells, plays a critical role in the urinary concentrating mechanisms. As a membrane protein, the function of UT-A1 transport activity relies on its presence in the plasma membrane. Therefore, UT-A1 successfully trafficking to the apical membrane of the polarized epithelial cells is crucial for the regulation of urea transport. This review summarizes the research progress of UT-A1 regulation over the past few years, specifically on the regulation of UT-A1 membrane trafficking by lipid rafts, N-linked glycosylation and a group of accessory proteins.     

The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO3− by the Porcine Colon

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L⁻¹ cinnamaldehyde induced increases in short circuit current (ΔI_sc) and conductance (ΔG_t) across the colon that were higher than those across the jejunum or after 1 mmol·L⁻¹ thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L⁻¹ quinidine or the bilateral replacement of 120 Na⁺, 120 Cl⁻ or 25 HCO₃⁻ reduced ΔG_t, while the removal of Ca²⁺ enhanced ΔG_t with ΔI_sc numerically higher. ΔI_sc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na⁺ or 25 HCO₃⁻. The removal of 120 Cl⁻ had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO₃⁻ via apical CFTR and basolateral Na⁺-HCO₃⁻ cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.