Spatiotemporal Changes of Tissue Glycans Depending on Localization in Cardiac Aging

Heart failure is caused by various factors, making its underlying pathogenic mechanisms difficult to identify, and tends to worsen over time. Early diagnosis of cardiovascular disease is the key for treatment to promote healthy life. To detect the structural and functional molecular changes associated with cardiovascular disease, we focused on glycans, which reflect the type and state of cells. We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging using an evanescent-filed lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. The glycan profiles in the left ventricle showed differences between the luminal side (medial) and the wall side (lateral) region. The former area was characterized by the presence of sialic acid residues.Moreover, age-related changes in glycan profiles were observed earlier in the medial region. The difference in the age-related decrease of a-galactose stained with griffonia simplicifolia lectin-IB4 in different region of the leftventricle suggested spatiotemporal changes in microvessels. The glycan profile, which retains diverse glycan structures, is supported by many cell populations and maintains cardiac function. Glycan localization and changes are expected to be developed as a marker of the signs and symptoms of heart failure in the future.

Visualizing Muscle Sialic Acid Expression in the GNED207VTgGne-/- Cmah-/- Model of GNE Myopathy: A Comparison of Dietary and Gene Therapy Approaches

Background: GNE myopathy (GNEM) is a rare, adult-onset, inclusion body myopathy that results from partial loss of function mutations in the GNE gene. GNE encodes UDP-GlcNAc epimerase/Mannose-6 kinase, a protein with two enzymatic activities that comprise the committed step in biosynthesis of sialic acid (SA), an essential glycan that appears on the terminal positions of many extracellular oligosaccharide chains. These GNE mutations can cause a reduction of SA in many tissues, although pathology is restricted to skeletal muscles through a poorly understood mechanism. Objective: Despite recent advances in the field, it remains unclear which therapeutic avenue is most promising for the restoration of SA level in skeletal muscle affected by GNEM. Our objective was to assess dietary and gene therapy strategies for GNEM in Cmah-deficient GNED207VTgGne-/- mice, a model that allows for the visualization of orally delivered N-glycolylneuraminic acid (Neu5Gc), one of the two predominant SA forms in muscle. Methods: Methods included in situ physiology studies of the tibialis anterior muscle, studies of ambulation and limb grip strength, and muscle staining using MAA, SNA, and anti-Neu5Gc antibody, along with qPCR, qRT-PCR, western blot, and HPLC studies to assess virally introduced DNA, GNE gene expression, GNE protein expression, and SA expression. Results: We found that a diet enriched in Neu5Gc-containing glycoproteins had no impact on Neu5Gc immunostaining in muscles of GNEM model mice. Delivery of a single high dose oral Neu5Gc therapy, however, did increase Neu5Gc immunostaining, though to levels below those found in wild type mice. Delivery of a single dose of GNE gene therapy using a recombinant Adeno Associated Virus (rAAV) vector with a liver-specific or a muscle-specific promoter both caused increased muscle Neu5Gc immunostaining that exceeded that seen with single dose monosaccharide therapy. Conclusions: Our findings indicate that dietary loading of Neu5Gc-containing glycoproteins is not effective in increasing muscle Neu5Gc expression, while single dose oral Neu5Gc monosaccharide or GNE gene therapy are. Neu5Gc immunostaining, however, showed greater changes than did lectin staining or HPLC analysis. Taken together, these results suggest that Neu5Gc immunostaining may be more sensitive technique to follow SA expression than other more commonly used methods and that liver expression of GNE may contribute overall muscle SA content.

Microtubule associated protein 4 (MAP4) phosphorylation reduces cardiac microvascular density through NLRP3-related pyroptosis

Phosphorylation of MAP4 (p-MAP4) causes cardiac remodeling, with the cardiac microvascular endothelium being considered a vital mediator of this process. In the current study, we investigated the mechanism underlying p-MAP4 influences on cardiac microvascular density. We firstly confirmed elevated MAP4 phosphorylation in the myocardium of MAP4 knock-in (KI) mice. When compared with the corresponding control group, we detected the decreased expression of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium of MAP4 KI mice, accompanied by a reduced plasma concentration of VEGF. Moreover, we observed apoptosis and mitochondrial disruption in the cardiac microvascular endothelium of MAP4 KI animals. Consistently, we noted a decreased cardiac microvascular density, measured by CD31 and lectin staining, in MAP4 KI mice. To explore the underlying mechanism, we targeted the NLRP3-related pyroptosis and found increased expression of the corresponding proteins, including NLRP3, ASC, mature IL-1β, IL-18, and GSDMD-N in the myocardium of MAP4 KI mice. Furthermore, we utilized a MAP4 (Glu) adenovirus to mimic cellular p-MAP4. After incubating HUVECs with MAP4 (Glu) adenovirus, the angiogenic ability was inhibited, and NLRP3-related pyroptosis were significantly activated. Moreover, both cytotoxicity and PI signal were upregulated by the MAP4 (Glu) adenovirus. Finally, NLRP3 inflammasome blockage alleviated the inhibited angiogenic ability induced by MAP4 (Glu) adenovirus. These results demonstrated that p-MAP4 reduced cardiac microvascular density by activating NLRP3-related pyroptosis in both young and aged mice. We thus managed to provide clues explaining MAP4 phosphorylation-induced cardiac remodeling and enriched current knowledge regarding the role of MAP4.

Lectin Staining of Microvascular Glycocalyx in Microfluidic Cancer Cell Extravasation Assays

The endothelial glycocalyx forms the inner-most lining of human microvasculature. It ensures the physiological function of blood vessels and plays a crucial role in the occurrence and progression of microvascular diseases. The present communication aims to highlight the usefulness of high-resolution imaging of lectin (Bandeiraea Simplicifolia) stained endothelial glycocalyx in 3-dimensional microfluidic cell cultures. The microfluidic system allowed visualizing cancer cell extravasation, which is a key event in metastasis formation in cancer pathologies. In brief, microvascular networks were created through spontaneous vasculogenesis. This occurred from 3 dimensional (3D) suspensions of human umbilical vein endothelial cells (HUVECs) in hydrogels confined within microfluidic devices. Extravasation of MDA-MB-231 breast cancer cells from perfusable endothelial lumens was observed with confocal imaging of lectin-stained microvascular networks. The present work provides guidance towards optimizing the methodology used to elucidate the role of the endothelial glycocalyx during cancer cell extravasation. In particular, a high-resolution view of the endothelial glycocalyx at the site of extravasation is presented. The occurrence of glycocalyx defects is well aligned with the contemporary notion in the field that glycocalyx shedding precedes cancer cell extravasation.

Nucleating biofilms for biotechnology using synthetic polymers

<p>This project is on developing robust biocatalysts in the form of enzymes expressed in biofilms. The aim is to design polymer scaffolds onto which bacteria adhere in a controlled manner, to form biofilms that can be used in biotechnology. Poly(acryloyl hydrazide) has been chosen as the polymer scaffold, due to easy synthesis and post-polymerization modification resulting in highly functional polymers<sup>1</sup> that are predicted to interact and cluster bacteria together. In this study poly(acryloyl hydrazide) with a range of hydrophobic functionalities were used to cluster and interact with two biofilm forming isogenic E.coli K-12 strains; PHL644 containing a point mutation ompR234 resulting in the overexpression of curli (a biofilm adhesin) and its parental wild type MC4100, with data showing correlations between polymer hydrophobicity, bacterial clustering and biofilm intensity in both strains. </p> <p>Data suggests these polymer induced clusters go on to develop many of the traits of a biofilm; crystal violet staining, lectin staining, and the use of reporter genes have confirmed the presence of extracellular polymeric substances, with their expression levels being directly linked to polymer hydrophobicity. Furthermore our polymers are able to boost biofilm intensity of MC4100 to the levels of PHL644, thereby opening up a potential avenue for non-biofilm forming but industrially relevant strains to experience the benefits of being in biofilm form (robustness, chemical and mechanical resistance).</p> <p>Biocatalytic ability of our polymer induced biofilms has also been tested, again with polymer properties dictating biotransformation yields.</p> <p>Finally, preliminary results have shown that we are able to selectively disperse our polymer induced biofilms, and then re-induce them by simply altering polymer properties in situ, providing a potentially useful reversible platform.</p> <ol> <li><strong>Polym. Chem.</strong>, 2017,<strong>8</strong>, 4576-4584</li> </ol>

Abstract TP96: Neuroprotective Effects of IGF-1 in Middle-Aged Females Can Be Replicated by Antagomirs to microRNA (miR)-92b and miR-33a

Background: Our previous studies show that intracerebroventricular (ICV) delivery of Insulin-like growth factor (IGF)-1 reduces MCAo-induced infarction in middle-aged female rats. Analysis of ischemic tissue at 4h showed that IGF1 treatment significantly reduced the expression of a group of miRNAs. To determine if inhibiting these miRNAs could replicate IGF-1-mediated neuroprotection, we examined the effects of antagomirs to miR-33a (A-mir33a) and miR-92b (A-mir92b) in in vivo and in vitro models of ischemia. Methods: Middle-aged (12 mo) acyclic female rats were subject to intraluminal MCAo for 90 min followed by reperfusion. Animals received a single tail vein injection of A-miR-33a, A-miR-92b, A-miR-33a+A-miR-92b or scrambled oligos (7ug/kg bwt) after 4h of reperfusion. We evaluated infarct volume (TTC staining) at 2d post stroke and behavioral recovery using neurological scores and adhesive removal test (ART). In parallel, human brain endothelial cell cultures were exposed to oxygen-glucose deprivation (OGD) in the presence of A-mir-33a, A-mir-92b or scrambled oligos. The integrity of the cell monolayer was assessed by tomato-lectin staining and cell death was assayed by LDH. Results: Single injection of A-mir92b during the acute stroke phase reduced infarct volume (p<0.04) and improved sensorimotor behavior (p<0.04) while A-mir33a treated animals were not different from scrambled control. However, the combination of A-mir-33a+A-mir-92b resulted in significantly smaller infarct volumes (p<0.04), less motor impairment (neurological score; p<0.02) and sensorimotor deficits (ART; p<0.05). In vitro studies showed that neither of the antagomirs reduced media LDH under OGD. As reported previously, OGD caused cell retraction and A-mir33a notably preserved the cell geometry (continuous lectin staining) to maintain the monolayer integrity and was similar to the normoxic control. Conclusions: IGF-1 regulated microRNAs appear to modulate specific aspects of stroke recovery. IV delivery of a combination of miRNA inhibitors may replicate the neuroprotective actions of IGF-1, thus providing a safer alternate to ICV delivery of IGF-1. Supported by R56 NS074895 and RF1 AG04218906