Wheat Germ Agglutinin Research Articles

Effect of Resistin on the Heart Structure of Mouse

Background: In recent years, relevant studies had shown that a large number of adipokines were involved in energy homeostasis in the body, regulating the balance of glucose and lipid metabolism. Resistin was a special type of adipose factor and its discovery and related studies had shown a close relationship between resistin and heart disease. However, there was relatively little research on the structure of heart tissue, so we conducted this present study. Methods: In order to study the effect of resistin on the heart structure of mice, 20 healthy C57BL/6 mice were randomly divided into experimental and control groups(n=10). The mice of the experimental group were injected with 20 ìl/d of resistin solution through the tail vein. The mice of the control group were injected with the same volume of double distilled water. HandE staining, Wheat Germ Agglutinin (WGA) staining, Masson’s trichrome staining and electron microscopy were used to observe the changes of microscopic structure and ultrastructure of mouse cardiomyocytes. Result: The results of HandE staining showed that the myocardial cells in the experimental group were disordered, the cytoplasm was loosely distributed and the gap between adjacent cells increased. WGA staining showed that the fluorescence green intensity of the mice in the experimental group was higherthan that of the control group. The myocardial cell membrane thickened, the average cross-sectional area of cardiomyocytes was larger than that of the control group. The results of the Masson staining showed that the staining of the cytoplasm of myocardial cells in the experimental group was significantly shallow and the blue structure was significantly increased. The electron microscopy showed that the number of mitochondria decreased and the mitochondria lightly dissolved or disappear. This study confirmed that resistin could change the content of collagen fibers and myofibrils in myocardial cells, induce cardiac hypertrophy and cause certain damage to mitochondria of cardiomyocytes. It was speculated that it could affect the physiological function of cardiomyocytes to some extent.

The counterbalance of endothelial glycocalyx and high wall shear stress to low-density lipoprotein concentration polarization in mouse ear skin arterioles

Background and aimsAtherosclerosis preferentially occurs at regions in arterial branching, curvature, and stenosis, which may be explained by the geometric predilection of low-density lipoprotein (LDL) concentration polarization that has been investigated in major arteries in previous studies. Whether this also happens in arterioles remains unknown. MethodsHerein, a radially non-uniform distribution of LDL particles and a heterogeneous endothelial glycocalyx layer in the mouse ear arterioles, as shown by fluorescein isothiocyanate labeled wheat germ agglutinin (WGA-FITC), were successfully observed by a non-invasive two-photon laser-scanning microscopy (TPLSM) technique. The stagnant film theory was applied as the fitting function to evaluate LDL concentration polarization in arterioles. ResultsThe concentration polarization rate (CPR, the ratio of the number of polarized cases to that of total cases) in the inner walls of curved and branched arterioles was 22% and 31% higher than the outer counterparts, respectively. Results from the binary logistic regression and multiple linear regression analysis showed that endothelial glycocalyx thickness increases CPR and the thickness of the concentration polarization layer (CPL). Flow field computation indicates no obvious disturbances or vortex in modeled arterioles with different geometries and the mean wall shear stress is about 7.7–9.0 Pa. ConclusionsThese findings suggest a geometric predilection of LDL concentration polarization in arterioles for the first time, and the existence of an endothelial glycocalyx, acting together with a relatively high wall shear stress in arterioles, may explain to some extent why atherosclerosis rarely occurs in these regions.

Vasculature in the mouse colon and spatial relationships with the enteric nervous system, glia, and immune cells.

The distribution, morphology, and innervation of vasculature in different mouse colonic segments and layers, as well as spatial relationships of the vasculature with the enteric plexuses, glia, and macrophages are far from being complete. The vessels in the adult mouse colon were stained by the cardiovascular perfusion of wheat germ agglutinin (WGA)-Alexa Fluor 448 and by CD31 immunoreactivity. Nerve fibers, enteric glia, and macrophages were immunostained in the WGA-perfused colon. The blood vessels entered from the mesentery to the submucosa and branched into the capillary networks in the mucosa and muscularis externa. The capillary net formed anastomosed rings at the orifices of mucosa crypts, and the capillary rings surrounded the crypts individually in the proximal colon and more than two crypts in the distal colon. Microvessels in the muscularis externa with myenteric plexus were less dense than in the mucosa and formed loops. In the circular smooth muscle layer, microvessels were distributed in the proximal, but not the distal colon. Capillaries did not enter the enteric ganglia. There were no significant differences in microvascular volume per tissue volume between the proximal and distal colon either in the mucosa or muscularis externa containing the myenteric plexus. PGP9.5-, tyrosine hydroxylase-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers were distributed along the vessels in the submucosa. In the mucosa, PGP9.5-, CGRP-, and vasoactive intestinal peptide (VIP)-immunoreactive nerves terminated close to the capillary rings, while cells and processes labeled by S100B and glial fibrillary acidic protein were distributed mainly in the lamina propria and lower portion of the mucosa. Dense Iba1 immunoreactive macrophages were closely adjacent to the mucosal capillary rings. There were a few macrophages, but no glia in apposition to microvessels in the submucosa and muscularis externa. In conclusion, in the mouse colon, (1) the differences in vasculature between the proximal and distal colon were associated with the morphology, but not the microvascular amount per tissue volume in the mucosa and muscle layers; (2) the colonic mucosa contained significantly more microvessels than the muscularis externa; and (3) there were more CGRP and VIP nerve fibers found close to microvessels in the mucosa and submucosa than in the muscle layers.

Heart failure, female sex and atrial fibrillation are the main drivers of atrial cardiomyopathy in cardiac surgery patients: results from the CATCH ME consortium

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): CATCH ME: Characterizing Atrial fibrillation by Translating its Causes into Health Modifiers in the Elderly Background Atrial cardiomyopathy is emerging as independent prognostic factor in cardiovascular disease. Fibrotic remodeling, cardiomyocyte hypertrophy and a reduction in capillary density are histological hallmarks of atrial cardiomyopathy. The contribution of etiological factors to atrial cardiomyopathy has not been robustly quantified. Purpose To quantify the relation between histological features of atrial cardiomyopathy and the clinical profile of patients. Methods We examined left (LA, n=91) and right (RA, n=75) atrial appendages sampled from a European cohort of patients undergoing cardiac surgery. Quantification of histological cardiomyopathy features was performed a recently developed and validated imaging analysis algorithm following myocardial triple staining with wheat germ agglutinin (WGA), CD31 and vimentin. The contributions of AF, heart failure (HF), sex, age, and 4 principal components accounting for confounding clinical characteristics to over-all and endomysial fibrosis were determined in a multivariate model. K-means clustering of 6 atrial histological features was performed to identify different types of remodeling. Results LA samples showed less fibrosis (p<0.001), a higher capillary density (p<0.05), a smaller capillary size (p<0.05), and larger cardiomyocytes (p<0.01) than RA samples. In both LA and RA, persistent AF was associated with endomysial fibrosis, while over-all fibrosis was not. Men had larger cardiomyocytes (LAA), while women had a higher degree of endomysial fibrosis (LA). Heart failure patients had more endomysial and over-all fibrosis (LA), a higher capillary density (LA) and capillary size (LA/RA). Samples clustered into 2 distinct clusters. One cluster showed fibrotic cardiomyopathy, with more endomysial (p<0.001) and overall fibrosis (p<0.001), more fibroblasts (p<0,001) and a higher capillary density (p<0.001). The other cluster showed hypertrophic cardiomyopathy, with hypertrophic cardiomyocytes (p<0.001). Patients with fibrotic cardiomyopathy were more often female (LA and RA, p<0.003) and had more often persistent AF (LA, p=0.031) or heart failure (LA, p<0.001). Patients with hypertrophic cardiomyopathy were more often male (LA and RA, p<0.003). Conclusions Fibrotic cardiomyopathy is associated with female sex, persistent AF and heart failure while hypertrophic cardiomyopathy more often occurs in men. More research is needed to establish whether treatment of AF and heart failure prevents the development of atrial cardiomyopathy.

Bladder Cancer Cells Interaction with Lectin-Coated Surfaces under Static and Flow Conditions.

Aberrant expression of glycans, i.e., oligosaccharide moiety covalently attached to proteins or lipids, is characteristic of various cancers, including urothelial ones. The binding of lectins to glycans is classified as molecular recognition, which makes lectins a strong tool for understanding their role in developing diseases. Here, we present a quantitative approach to tracing glycan-lectin interactions in cells, from the initial to the steady phase of adhesion. The cell adhesion was measured between urothelial cell lines (non-malignant HCV29 and carcinoma HT1376 and T24 cells) and lectin-coated surfaces. Depending on the timescale, single-cell force spectroscopy, and adhesion assays conducted in static and flow conditions were applied. The obtained results reveal that the adhesion of urothelial cells to two specific lectins, i.e., phytohemagglutinin-L and wheat germ agglutinin, was specific and selective. Thus, these lectins can be applied to selectively capture, identify, and differentiate between cancer types in a label-free manner. These results open up the possibility of designing lectin-based biosensors for diagnostic or prognostic purposes and developing strategies for drug delivery that could target cancer-associated glycans.

High-mobility group box 1 (HMGB1) crosses the BBB bidirectionally

High-mobility group box 1 (HMGB1) is a ubiquitous protein that regulates transcription in the nucleus, and is an endogenous damage-associated molecular pattern molecule that activates the innate immune system. HMGB1 activates the TLR4 and RAGE recepto, inducing downstream signals reminiscent of cytokines that have been found to cross the blood–brain barrier (BBB). Blood HMGB1 increases in stroke, sepsis, senescence, alcohol binge drinking and other conditions. Here, we examined the ability of HMGB1 radioactively labeled with iodine (I-HMGB1) to cross the BBB. We found that I-HMGB1 readily entered into mouse brain from the circulation with a unidirectional influx rate of 0.654 μl/g-min. All brain regions tested took up I-HMGB1; uptake was greatest by the olfactory bulb and least in the striatum. Transport was not reliably inhibited by unlabeled HMGB1 nor by inhibitors of TLR4, TLR2, RAGE, or CXCR4. Uptake was enhanced by co-injection of wheatgerm agglutinin, suggestive of involvement of absorptive transcytosis as a mechanism of transport. Induction of inflammation/neuroinflammation with lipopolysaccharide is known to increase blood HMGB1; we report here that brain transport is also increased by LPS-induced inflammation. Finally, we found that I-HMGB1 was also transported in the brain-to-blood direction, with both unlabeled HMGB1 or lipopolysaccharide increasing the transport rate. These results show that HMGB1 can bidirectionally cross the BBB and that those transport rates are enhanced by inflammation. Such transport provides a mechanism by which HMGB1 levels would impact neuroimmune signaling in both the brain and periphery.

Extending the use of biologics to mucous membranes by attachment of a binding domain

Biologics are almost exclusively administered systemically, but localized delivery is preferable as it minimizes off-target exposure and allows more aggressive treatments. Topical application of biologics to epithelia is generally ineffective because most are covered with fluids and biologics are washed out too quickly to have significant therapeutic effects. Here we explore the idea that attaching a binding domain can serve as an “anchor” to extend the residency time of biologics on wet epithelia, allowing their effective use even with infrequent applications. We use topical application to the ocular surface as a challenging test since foreign substances are washed out especially efficiently by tear flow and blinking. Our results demonstrate that conjugation of antibodies to wheat germ agglutinin, which binds GlcNAc and sialic acid that are ubiquitously present in tissues, increases their half-life 350-fold upon application to the ocular surface in a mouse model of dry eye, a common and onerous disease in humans. Importantly, antibodies to IL-17A, IL-23, and IL-1β conjugated to the agglutinin reduces manifestations of dry eye, even when applied just once daily. In contrast, unconjugated antibodies are ineffective. Attaching an anchor to biologics is a simple means to overcome washout and to extend their therapeutic use.

MP-453085-5 ATRIAL FIBRILLATION, FEMALE SEX AND HEART HAILURE ARE THE MAIN DRIVERS OF ATRIAL CARDIOMYOPATHY IN CARDIAC SURGERY PATIENTS: RESULTS FROM THE CATCH ME CONSORTIUM

Atrial cardiomyopathy is emerging as independent prognostic factor in cardiovascular disease. Fibrotic remodeling, cardiomyocyte hypertrophy and a reduction in capillary density are histological hallmarks of atrial cardiomyopathy. The contribution of etiological factors to atrial cardiomyopathy has not been robustly quantified. To quantify the relation between histological features of atrial cardiomyopathy and the clinical profile of patients. We examined left (LAA, n=91) and right (RAA, n=75) atrial appendages sampled from a European cohort of patients undergoing cardiac surgery. Quantification of histological cardiomyopathy features was performed using a recently developed and validated imaging analysis algorithm following myocardial triple staining with wheat germ agglutinin (WGA), CD31 and vimentin. The contributions of AF, heart failure (HF), sex, age, and 4 principal components accounting for confounding clinical characteristics to over-all and endomysial fibrosis were determined in a multivariate model. K-means clustering of 6 atrial histological features was performed to identify different types of remodeling. LAA samples showed less fibrosis (p<0.001), a higher capillary density (p<0.05), a smaller capillary size (p<0.05), and larger cardiomyocytes (p<0.01) than RAA samples. In both LAA and RAA, persistent AF was associated with endomysial fibrosis, while over-all fibrosis was not. Men had larger cardiomyocytes (LAA), while women had a higher degree of endomysial fibrosis (LAA). Heart failure patients had more endomysial and over-all fibrosis (LAA), a higher capillary density (LAA) and capillary size (LAA/RAA). Samples clustered into 2 distinct clusters. One cluster showed fibrotic cardiomyopathy, with more endomysial (p<0.001) and overall fibrosis (p<0.001), more fibroblasts (p<0,001) and a higher capillary density (p<0.001). The other cluster showed hypertrophic cardiomyopathy, with hypertrophic cardiomyocytes (p<0.001). Patients with fibrotic cardiomyopathy were more often female (LAA and RA, p<0.003) and had more often persistent AF (LAA, p=0.031) or heart failure (LAA, p<0.001). Patients with hypertrophic cardiomyopathy were more often male (LAA and RAA, p<0.003). Fibrotic cardiomyopathy is associated with female sex, persistent AF and heart failure while hypertrophic cardiomyopathy more often occurs in men. More research is needed to establish whether treatment of AF and heart failure prevents the development of atrial cardiomyopathy.

Benefice of the stimulation of O-GlcNAcylation on sodium potassium pump during hemorrhagic shock

Hemorrhagic shock (HS) is the consequence of an important loss in blood volume leading to an insufficient delivery in oxygen. HS causes metabolic changes, multi-organ dysfunction and inflammation. The acute kidney injury related to HS is associated with hyperkalemia mainly through an alteration of Na/K ATPase pump. In a previous O-GlcNAcylomic study, we have identified Na/K ATPase as potentially O-GlcNAcylated protein. O-GlcNAcylation is a post-translational modification with a putative beneficial effect in shock situation. This study aims at evaluating if Na/K ATPase is O-GlcNAcylated and its potential impact during the early phase of hemorrhagic shock. Wistar rats were subjected to a HS protocol induced via exsanguination and then randomly treated or not with NButGT (10 mg/kg), a O-GlcNAcase inhibitor, to increase O-GlcNAc levels. Blood pressure and heart rate were monitored throughout the experiment. Blood was collected from the animals to perform a complete blood count and a blood gas analysis. Kidney was immediately frozen for histological and biochemical studies. Immunoprecipitation and Wheat Germ Agglutinin (WGA) based lectin affinity gel electrophoresis were used to study the O-GlcNAcylation of Na/K ATPase. NButGT increased by 2 O-GlcNAc levels in heart and kidney. This increase in O-GlcNAc restored mean arterial pressure (MAP) (P < 0.01) and bicarbonates (P < 0.01). In treated group, kalemia (Sham: 4.2 ± 0.1; HS: 4.7 ± 0.1; NButGT: 4.3 ± 0.1 mmol/L; P < 0.05) and natremia (Sham: 141.5 ± 0.4; HS: 139.8 ± 0.5; NButGT: 141.9 ± 0.4 mmol/L; P < 0.01) were restored. The study of the O-GlcNAcylation of the Na/K ATPase by immunoprecipitation and WGA gel validated its O-GlcNAcylation. Histological analysis of kidney revealed that HS reduced NA/K ATPase expression during HS and treatment by NButGT restored it (Sham: 15.1 ± 1.1; HS: 10.3 ± 0.6; NButGT: 13.78 ± 1.1; % area; P < 0.05). Our results demonstrate that increase O-GlcNAcylation during hemorrhagic shock restored MAP and reduced markers of metabolic acidosis. In our study, animal treatment with NButGT to increase O-GlcNAc levels, restored natremia and kalemia. This effect is associated with an increase in Na/K ATPase expression. To our knowledge, our study validates the O-GlcNAcylation of the Na/K ATPase. With regard to our results, now it will be relevant to study the effect of O-GlcNAcylation on the activity and localisation.

Abstract 312: Diosmin Enhances Endothelial Glycocalyx Integrity And Reverses Endothelial Dysfunction In Injured Blood Vessels

Objective: This study determined if diosmin, a flavone glycoside of diosmetin, can repair damaged extracellular endothelial glycocalyx (GCX) (readouts: GCX labels including wheat germ agglutinin (WGA), hyaluronic acid (HA) binding protein, and core protein syndecan-1 (SDC1) antibody). Additionally, the study wanted to identify if diosmin can reverse endothelial dysfunction by improving vascular tone (readouts: endothelial nitric oxide synthase expression (eNOS), vessel wall thickness, and vessel lumen diameter) and vascular permeability (readouts: inflammatory cell uptake). Methods: In a mouse, acute disturbed blood flow pattern was induced by ligating three of the four left carotid artery (LCA) branches. This partial LCA ligation mouse model has shown to induce rapid endothelial dysfunction and loss of endothelial GCX. The right carotid artery (RCA) was the designated control for each mouse, as no surgical intervention occurred. Diosmin (50 mg/kg) was administered daily either orally or intraperitoneally for 7 days, 72 hours after the ligation surgery. One week after the first treatment dose, mice were sacrificed via exsanguination, and tissue was collected and fixed for further processing. Results: Diosmin enhanced endothelial GCX coverage and thickness after one week of treatment, as demonstrated by increased fluorescence of WGA, HA binding protein, and SDC1 antibody label on the diosmin-treated ligated LCA wall, compared to untreated tissue. Furthermore, diosmin also appeared to improve vascular tone as demonstrated by the increase in percent of total eNOS that is activated (p-eNOS/total eNOS) in diosmin-treated ligated LCA, compared to the untreated LCA. Interestingly, diosmin had limited effect on vessel wall thickness and vessel diameter. Finally, diosmin demonstrated attenuation of vessel inflammation by impeding macrophage (CD68 + ) uptake within ligated LCA walls. Conclusion: The novelty of our findings is that diosmin enhances endothelial GCX integrity. This study also demonstrated that diosmin can improve endothelium functionality after injury by decreasing inflammation and improving vascular tone. This work can lead to a new option for endothelial GCX restoration and endothelial-dependent aspects of vascular disease.

Inducible deletion of endothelial cell EfnB2 attenuates neuromuscular regeneration in mouse skeletal muscle

Background: Acute injury of skeletal muscle disrupts myofibers, microvessels and neuromuscular junctions (NMJs). While myofiber regeneration is well-characterized, little is known of concurrent events in microvessels and motor nerves following injury. Endothelial cell [EC] ephrin B2 [EfnB2] is required for sprouting angiogenesis during embryonic development, for promoting vascularization of tumors in the adult, and for microvascular-dependent guidance of axon regeneration following nerve resection. Hypothesis: We hypothesized that following acute injury to skeletal muscle 1) EC EfnB2 is required for restoring microvascular volume; and 2) loss of EC EfnB2 would impair recovery of neuromuscular structure and function. Methods: Mice (male and female, age 3-5 months) were bred for EC-specific conditional knockout [CKO] of EfnB2 following tamoxifen injection; noninjected CKO mice served as “wildtype” controls [WT]. Following injury by local injection of the myotoxin BaCl2, the gluteus maximus [GM] muscle was studied for microvascular analysis or the tibialis anterior [TA] muscle was evaluated for recovery of neuromuscular function. Mice were studied (n=4-6/group) at 3 timepoints for vascular studies (0, 5, and 10 days post injury [dpi]) and 2 timepoints for neuromuscular structure and function (0 and 10 dpi). Results: Intravascular staining with fluorescent wheat germ agglutinin revealed a diminished microvascular area in the GM of CKO mice at 5 dpi vs. 10 dpi (means ± SE; CKO 5 dpi 35.5% ± 2.5 vs. CKO 10 dpi 49.8% ± 3.9, P&lt;0.01). No difference in vascular area was recorded in WT groups at 5 vs. 10 dpi (WT 5 dpi 45.5% ± 2.6 vs. WT 10 dpi 46.7% ± 2.2, P=0.99). Direct electrical stimulation of the TA resolved a reduction in maximal force at 10 dpi when compared to 0 dpi for both WT and CKO (WT 0 dpi 128.6 g ± 4.1 vs. WT 10 dpi 64.7 g ± 3.0, P&lt;0.0001) (CKO 0 dpi 112.7 g ± 5.0 vs. CKO 10 dpi 65.9 g ± 5.0, P&lt;0.0001). Indirect stimulation (sciatic nerve) resolved initial neuromuscular transmission failure (INMTF) reduced submaximal force in CKO but not WT (CKO 0 dpi 2.0 ± 4.3% vs. CKO 10 dpi -30.0 ± 6.5 %, P&lt;0.001), which correlated with perturbed recovery of NMJ innervation; (R2=0.328, P=0.003), endplate area; (R2=0.479, P=0.001), and nicotinic receptor clusters; (R2=0.207, P=0.025). Conclusion: Following acute injury of skeletal muscle in the adult mouse, loss of EfnB2 in ECs delays microvessel regeneration in the GM and disrupts the recovery of NMJ structure and function in the TA. Support: NIH grant F32 HL-152558 (NLJ), APS Postdoctoral Fellowship (ABM), NIH LRP (ABM), Margaret Proctor Mulligan Fellowship, MU (NLJ, SSS), NIH R01 AR078045 (DDC), School of Medicine Development Award, MU (SSS), NIH MERIT Award R37 HL-041026 (SSS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prolonged intermittent hypoxia causes mitochondrial dysfunction due to O-GlcNacylation of DRP1 in models of COPD (chronic obstructive pulmonary disease)

Background: Sarcopenia, or skeletal muscle loss, is common complication in COPD and contributes to adverse clinical outcomes including mortality. As the disease progresses, a significant proportion of COPD patients develop nocturnal hypoxemia which causes tissue-level hypoxia in the skeletal muscle which we have termed prolonged intermittent hypoxia (PIH). We have demonstrated sarcopenia, mitochondrial dysfunction, and dysregulated hypoxia-inducible factor 1-α (HIF1-α) responses both in vitro and in vivo in response to PIH. We also noted multiple transcriptional targets of HIF1-α were upregulated in the glycolytic pathway. We hypothesized that PIH upregulates the Hexosamine Biosynthetic (HB) pathway, a branch of glycolysis responsible for post-translational glycosylation of proteins, to cause O-GlcNAcylation of DRP1 leading to increased mitochondrial fission, fragmented mitochondrial networks, mitochondrial dysfunction, and sarcopenia. Methods: Our in vitro model of PIH exposed differentiated, murine C2C12 myotubes to 16h normoxia/8h hypoxia [1% oxygen] for 3 days, while our in vivo model exposed C57BL/6 mice to 12h normoxia/12h hypoxia [10% oxygen] for 21 days. Protein extraction and immunoblots were performed to probe for enzymes in the HB, mTORC1 pathways, O-GlcNAcylation, and puromycin incorporation. Imaging of mitochondrial structure was determined by staining with MitoTracker orange and electron microscopy. Immunoprecipitation and detection of Drp1 O-GlcNAcylation was performed by incubating protein lysates with Wheat Germ Agglutinin coated Agarose beads. To determine responses to inhibition of O-GlcNAcylation, we performed CRISPR-Cas9 RNP deletion of GFAT, the first enzyme in the HB pathway. Results: We found significant increase in O-GlcNAcylated proteins in our PIH models . This was accompanied by increased expression of HIF1-α, mitochondrial dysfunction, and upregulated enzymes in the HB pathway. O-GlcNAcylated modification of DRP1 was significantly upregulated due to PIH in vitro. GFAT KO myotubes demonstrated significant reductions in global O-GlcNAcylation of proteins and O-GlcNAcylated Drp1 with PIH. Interestingly, protein synthesis was rescued in GFAT KO myotubes as determined by puromycin incorporation, restoration of mTORC1 signaling, and reversal of a sarcopenic phenotype. Conclusion: Our findings demonstrate a potential mechanism for mitochondrial dysfunction and sarcopenia due to PIH which may be translationally relevant for patients with COPD. Future studies focused on O-GlcNAcylated modification of DRP1 and the functional consequences are needed to identify novel therapeutic targets to reverse sarcopenia in COPD. Supported in part by: NIH RO1 GM119174; RO1 DK113196; P50 AA024333; RO1 AA021890; 3U01AA026976 - 03S1; UO1 AA 026976; R56HL141744;UO1 DK061732; 5U01DK062470-17S2; R21 AR 071046; Howard and Helen Trevey Endowment; (SD); K12 HL141952 (AA) and the American College of Gastroenterology Clinical Research Award and NIH KO8 AAAA028794 (NW). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Human serum albumin nanoparticles as a versatile vehicle for targeted delivery of antibiotics to combat bacterial infections

Urinary tract infections (UTIs) are among the most common bacterial infections. Despite a wide range of therapeutic options, treatment success is compromised by the efficient mechanism of tissue colonization of uropathogenic Escherichia coli. In advanced drug delivery systems, a similar, glycan-mediated targeting mechanism may be realized by conjugating the drug to a plant lectin, like wheat germ agglutinin (WGA).We introduce a drug delivery vehicle consisting of human serum albumin as nanoparticle shell, olive oil as core component, the active pharmaceutical ingredients (API) trimethoprim and rifampicin as well as WGA to facilitate cellular internalization. When WGA was embedded into the proteinaceous particle shell, cell binding studies revealed up to 60 % higher cell binding potential. Additionally, nanoparticles showed a good efficacy against gram-negative just as against gram-positive bacteria.The combination of the promising cell-associative properties and the proven antimicrobial potential might lead to an improved efficacy of advanced treatment of UTIs.

The infectious salmon anemia virus esterase prunes erythrocyte surfaces in infected Atlantic salmon and exposes terminal sialic acids to lectin recognition.

Many sialic acid-binding viruses express a receptor-destroying enzyme (RDE) that removes the virus-targeted receptor and limits viral interactions with the host cell surface. Despite a growing appreciation of how the viral RDE promotes viral fitness, little is known about its direct effects on the host. Infectious salmon anemia virus (ISAV) attaches to 4-O-acetylated sialic acids on Atlantic salmon epithelial, endothelial, and red blood cell surfaces. ISAV receptor binding and destruction are effectuated by the same molecule, the haemagglutinin esterase (HE). We recently discovered a global loss of vascular 4-O-acetylated sialic acids in ISAV-infected fish. The loss correlated with the expression of viral proteins, giving rise to the hypothesis that it was mediated by the HE. Here, we report that the ISAV receptor is also progressively lost from circulating erythrocytes in infected fish. Furthermore, salmon erythrocytes exposed to ISAV ex vivo lost their capacity to bind new ISAV particles. The loss of ISAV binding was not associated with receptor saturation. Moreover, upon loss of the ISAV receptor, erythrocyte surfaces became more available to the lectin wheat germ agglutinin, suggesting a potential to alter interactions with endogenous lectins of similar specificity. The pruning of erythrocyte surfaces was inhibited by an antibody that prevented ISAV attachment. Furthermore, recombinant HE, but not an esterase-silenced mutant, was sufficient to induce the observed surface modulation. This links the ISAV-induced erythrocyte modulation to the hydrolytic activity of the HE and shows that the observed effects are not mediated by endogenous esterases. Our findings are the first to directly link a viral RDE to extensive cell surface modulation in infected individuals. This raises the questions of whether other sialic acid-binding viruses that express RDEs affect host cells to a similar extent, and if such RDE-mediated cell surface modulation influences host biological functions with relevance to viral disease.

EXOSOMES AND MICROVESICLES FROM ADIPOSE-DERIVED MESENCHYMAL STEM CELLS PROTECTS THE ENDOTHELIAL GLYCOCALYX FROM LPS INJURY.

Introduction: Endothelial glycocalyx damage occurs in numerous pathological conditions and results in endotheliopathy. Extracellular vesicles, including exosomes and microvesicles, isolated from adipose-derived mesenchymal stem cells (ASCs) have therapeutic potential in multiple disease states; however, their role in preventing glycocalyx shedding has not been defined. We hypothesized that ASC-derived exosomes and microvesicles would protect the endothelial glycocalyx from damage by LPS injury in cultured endothelial cells. Methods : Exosomes and microvesicles were collected from ASC conditioned media by centrifugation (10,000 g for microvesicles, 100,000 g for exosomes). Human umbilical vein endothelial cells (HUVECs) were exposed to 1 μg/mL lipopolysaccharide (LPS). LPS-injured cells (n = 578) were compared with HUVECS with concomitant LPS injury plus 1.0 μg/mL of exosomes (n = 540) or microvesicles (n = 510) for 24 hours. These two cohorts were compared with control HUVECs that received phosphate-buffered saline only (n = 786) and HUVECs exposed to exosomes (n = 505) or microvesicles (n = 500) alone. Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify EGX. Real-time quantitative reverse-transcription polymerase chain reaction was used on HUVECs cell lystate to quantify hyaluron synthase-1 (HAS1) expression. Results: Exosomes alone decreased endothelial glycocalyx staining intensity when compared with control (4.94 vs. 6.41 AU, P < 0.001), while microvesicles did not cause a change glycocalyx staining intensity (6.39 vs. 6.41, P = 0.99). LPS injury resulted in decreased glycocalyx intensity as compared with control (5.60 vs. 6.41, P < 0.001). Exosomes (6.85 vs. 5.60, P < 0.001) and microvesicles (6.35 vs. 5.60, P < 0.001) preserved endothelial glycocalyx staining intensity after LPS injury. HAS1 levels were found to be higher in the exosome (1.14 vs. 3.67 RE, P = 0.02) and microvesicle groups (1.14 vs. 3.59 RE, P = 0.02) when compared with LPS injury. Hyaluron synthase-2 and synthase-3 expressions were not different in the various experimental groups. Conclusions: Exosomes alone can damage the endothelial glycocalyx. However, in the presence of LPS injury, both exosomes and microvesicles protect the glycocalyx layer. This effect seems to be mediated by HAS1. Level of Evidence : Basic science study.

Simple and rapid co-freezing construction of SERS signal probes for the sensitive detection of pathogens

In this study, we proposed a rapid and sensitive surface-enhanced Raman scattering (SERS)-based biosensor for pathogen detection. The biosensor utilized wheat germ agglutinin (WGA)-modified raspberry-like Fe3O4@Au magnetic nanoparticles (RAuMNPs) as capture elements to enrich the target pathogen and used co-freezing aptamer/5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)/gold nanoparticles (AuNps) tags as the signal source. Compared to conventional AuNps tags, aptamer/DTNB tags manufactured by co-freezing were easy to prepare, highly stable and had a high affinity for the target bacteria. WGA-coated RAuMNPs captured Escherichia coli (E. coli) O157:H7 from real samples, such as human urine, serum, and skimmed milk, within 20 min under optimized conditions. The combined use of the co-freezing SERS tags and RAuMNPs-WGA significantly improved the analytical sensitivity of the proposed SERS platform, enabling detection as low as 8 cells/mL of target bacteria with a linear range of 10 to 106 cells/mL. Furthermore, the platform successfully detected DNA from difficult-to-culture or slow-growing pathogens, such as Mycobacterium tuberculosis. Overall, this sensitive and fast SERS platform has promising potential for accurate and efficient identification of pathogenic bacteria from cultures and various samples.

Wheat germ agglutinin conjugated chitosan nanoparticles for gemcitabine delivery in MCF-7 cells; synthesis, characterisation and in vitro cytotoxicity studies.

Numerous clinical trials indicated combination regimens containing gemcitabine could extend progression-free survival of breast cancer patients without increasing the incidence of serious adverse effects. Orally administered gemcitabine is being metabolized by enzymes present in intestinal cells rapidly; thereupon, the current study was aimed to preparing, optimizing, and evaluating cytotoxicity of wheat germ agglutinin conjugated gemcitabine-chitosan nanoparticles (WGA-Gem-CNPs) in MCF-7 and HEK293 cells and to determining their cellular uptake by Caco-2 cells. Gem-CNPs were prepared by Ionic Gelation method and optimum formulation was implied for WGA conjugation optimisation. Nanoparticles formation was approved by FTIR and DSC analyses; then particles were characterized by DLS and release profile was prepared. MTT assay was performed in MCF-7 and HEK293. Optimized Gem-CNPs and WGA-Gem-CNPs particle size were estimated as 126.6 ± 21.8 and 144.8 ± 36.1 nm, respectively. WGA conjugation efficacy was calculated as 50.98 ± 2.32 percent and encapsulation efficiency in WGA-Gem-CNPs was 69.44 ± 3.41 percent. Three-hour Caco-2 cellular uptake from Gem-CNPs and WGA-Gem-CNPs were estimated as averagely 3.5 and 4.5 folds higher than free drug, respectively. Gem-CNPs and WGA-Gem-CNPs reduced IC50 in MCF-7 cells by 2 and 2.5 folds, respectively; such decrease for HEK293 cells was as much as 2.4 and 6.3 folds, in same order. Demonstrated significant in vitro uptake of WGA-Gem-CNPs and cytotoxicity might be considered for more studies as a potential carrier for oral delivery of gemcitabine.

Cover Image

3D rendering of medullary ray sheath fibers surrounding tubular elements and glomeruli in an embryonic day (E)18.5 mouse Foxd1GC/+; COL5A1fl/fl kidney. Decellularized E18.5 kidney stained for type V collagen (green), type IV collagen (blue), and proteoglycans (wheat germ agglutinin, red) imaged using confocal microscopy. From: FOXD1 is required for 3D patterning of the kidney interstitial matrix; Lipp SN, Jacobson KR, Schwaderer AL, Hains DS, Calve S; Developmental Dynamics. 2023;252(4): 463-482. doi: 10.1002/dvdy.545

A Novel Preparation Technique for Human Nasal Respiratory Mucosa to Disclose Its Glycosylation Pattern for Bioadhesive Drug Delivery

To shed some light on glycotargeting as a potential strategy for nasal drug delivery, a reliable preparation method for human nasal mucosa samples and a tool to investigate the carbohydrate building blocks of the glycocalyx of the respiratory epithelium are required. Applying a simple experimental setup in a 96-well plate format together with a panel of six fluorescein-labeled lectins with different carbohydrate specificities allowed for the detection and quantification of accessible carbohydrates in the mucosa. As confirmed by binding experiments at 4 °C, both quantitatively by fluorimetry and qualitatively by microscopy, the binding of wheat germ agglutinin exceeded that of the others by 150% on average, indicating a high content of N-acetyl-D-glucosamine and sialic acid. Providing energy by raising the temperature to 37 °C revealed uptake of the carbohydrate-bound lectin into the cell. Moreover, repeated washing steps during the assay gave a slight hint as to the influence of mucus renewal on bioadhesive drug delivery. All in all, the experimental setup reported here for the first time is not only a suitable approach to estimating the basics and potential of nasal lectin-mediated drug delivery but also meets the needs for answering a broad variety of scientific questions involving the use of ex vivo tissue samples.

Silicate Ions Derived from Calcium Silicate Extract Decelerate Ang II-Induced Cardiac Remodeling.

Pathological cardiac hypertrophy is one of the main activators of heart failure. Currently, no drug can completely reverse or inhibit the development of pathological cardiac hypertrophy. To this end, we proposed a silicate ion therapy based on extract derived from calcium silicate (CS) bioceramics for the treatment of angiotensin II (Ang II) induced cardiac hypertrophy. In this study, the Ang II induced cardiac hypertrophy mouse model was established, and the silicate ion extract was injected to mice intravenously. The cardiac function was evaluated by using a high-resolution Vevo 3100 small animal ultrasound imaging system. Wheat germ Agglutinin, Fluo4-AM staining and immunofluorescent staining was conducted to assess the cardiac hypertrophy, intracellular calcium and angiogenesis of heart tissue, respectively. The in vitro results showed that silicate ions could inhibit the cell size of cardiomyocytes, reduce cardiac hypertrophic gene expression, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), decrease the content of intracellular calcium induced by Ang II. In vivo experiments in mice confirmed that intravenous injection of silicate ions could remarkably inhibit the cardiac hypertrophy and promote the formation of capillaries, further alleviating Ang II-induced cardiac function disorder. This study demonstrated that the released silicate ions from CS possessed potential value as a novel therapeutic strategy of pathological cardiac hypertrophy, which provided a new insight for clinical trials.