Hyaluronan Binding Research Articles

Automated smartphone-based system for measuring sperm viability, DNA fragmentation, and hyaluronic binding assay score.

The fundamental test for male infertility, semen analysis, is mostly a manually performed subjective and time-consuming process and the use of automated systems has been cost prohibitive. We have previously developed an inexpensive smartphone-based system for at-home male infertility screening through automatic and rapid measurement of sperm concentration and motility. Here, we assessed the feasibility of using a similar smartphone-based system for laboratory use in measuring: a) Hyaluronan Binding Assay (HBA) score, a quantitative score describing the sperm maturity and fertilization potential in a semen sample, b) sperm viability, which assesses sperm membrane integrity, and c) sperm DNA fragmentation that assesses the degree of DNA damage. There was good correlation between the manual analysis and smartphone-based analysis for the HBA score when the device was tested with 31 fresh, unprocessed human semen samples. The smartphone-based approach performed with an accuracy of 87% in sperm classification when the HBA score was set at manufacturer’s threshold of 80. Similarly, the sperm viability and DNA fragmentation tests were also shown to be compatible with the smartphone-based system when tested with 102 and 47 human semen samples, respectively.

Hyaluronan bound mature sperm count (HB-MaSC) is a more informative indicator of fertility than conventional sperm parameters: Correlations with Body Mass Index (BMI)

The relationship between overweight and male fertility is well studied, still the correlation of obesity and decreased sperm quality is a subject to debate. The widely used conventional spermatological examinations alone seem to be inadequate to assess fertilization potential. Hyaluronan Binding Assay (HBA®) is one of the available validated tests that allows the functional examination of sperm. Data of 72 male patients (mean age 33.9 (24–43) years) from infertile couples were analysed. Body Mass Index (BMI) determination, conventional semen analysis and HBA were performed. Additionally, a relatively new Hyaluronan Bound Matured Sperm Count (HB-MaSC) -index, first introduced by the authors in 2015, was calculated. This index reflects fertilization potential of sperm more precisely. With the increase of BMI, sperm count decreased significantly until about 25 kg/m2, above 25 kg/m2 no further decrease was observed, although sperm count remained permanently low. Greater body weight (in the 70–90 kg range) was observed to have a significant negative effect only on the progressive sperm motility. In addition to sperm concentration and motility, sperm fertilization potential is also negatively affected by obesity, but is irrespective of body weight, as evaluated using BMI + HB-MaSC linear regression analyses adjusted for age and weight. This correlation between male BMI and sperm fertilization potential – as opposed to the conventional correlations with sperm concentration or motility – appears to provide more helpful information in the identification of real capability for fertilization.

Sperm selection for assisted reproduction by prior hyaluronan binding: the HABSelect RCT

BackgroundSperm selection for intracytoplasmic sperm injection (ICSI) has traditionally relied on standardised methods of sperm processing combined with subjective sperm selection (motility/morphology). In 2012, live birth rates (LBRs) stood at ≈24% per cycle started (32% per cycle reaching embryo transfer).Objective(s)The main clinical objective was to determine the benefits of a hyaluronan (HA)-based sperm selection process for physiological intracytoplasmic sperm injection (PICSI). A parallel, mechanistic objective evaluated sperm chromatin integrity and the potential of PICSI to compensate for poor sperm quality.DesignA Phase III, parallel-arm, blinded randomised controlled trial (RCT) of efficacy of PICSI versus ICSI alongside mechanistic evaluation.SettingThe RCT ran from February 2014 to August 2016, involving NHS (n = 14) and private (n = 2) UK hospital or satellite clinics. Mechanistic work was conducted in three university-based research laboratories and a partnering small–medium enterprise from June 2015 to December 2017.ParticipantsCouples undergoing an ICSI procedure using freshly ejaculated sperm with female partners aged between 18 and 43 years and male partners aged between 18 and 55 years.InterventionHealth and Care Professions Council-registered embryologists used the Medicines and Healthcare products Regulatory Agency-registered (HA-coated) PICSI™ dish (Origio, Måløv, Denmark) to select a single sperm for injection. Control couples received standard care.Main outcome measuresClinical – the primary outcome was full-term live birth (≥ 37 weeks’ gestation). Secondary outcome measures were confirmed clinical pregnancy (CP), miscarriage following confirmation and preterm live birth (< 37 weeks’ gestation). Mechanistic – measurement models were designed for deoxyribonucleic acid (DNA) fragmentation, compaction and HA binding [HA binding score (HBS)].ResultsA total of 2772 couples were randomised and 2752 couples were included in the primary analysis (PICSI,n = 1371; and ICSI,n = 1381). Clinical – primary outcome: 379 out of 1381 (27.4% PICSI) and 346 out of 1371 (25.2% ICSI) couples who were randomised (up to 24 hours before treatment) into the trial achieved a term live birth ≥ 37 weeks’ gestation [odds ratio (OR) 1.12, 95% confidence interval (CI) 0.94 to 1.34;p = 0.18]. Subgroup analyses did not reveal differences in treatment effects for HBS, maternal age, previous miscarriage, follicle-stimulating hormone or anti-Müllerian hormone levels and paternal sperm concentrations. Secondary outcomes: CP was achieved for 487 out of 1382 (35.2% PICSI) and 491 out of 1375 (35.7%, ICSI) couples (OR 0.98, 95% CI 0.84 to 1.15;p = 0.80). Miscarriage affected 60 out of 1381 (4.3% PICSI) and 96 out of 1371 (7.0% ICSI) of couples (OR 0.61, 95% CI 0.43 to 0.84;p = 0.003). Preterm LBRs were 46 out of 1381 (3.3% PICSI) and 45 out of 1371 (3.3% ICSI) (OR 1.02, 95% CI 0.67 to 1.55;p = 0.94). Mechanistic: in the subset of samples examined, HBS correlated with sperm motility, concentration, fertilisation rate and DNA fragmentation. Sperm DNA compaction was weakly associated with clinical pregnancy rates (CPRs), but neither HBS nor DNA fragmentation was predictive of any clinical outcome.LimitationsEmbryologists were not blinded and limited data were available from poorer samples and non-random sample selection in the mechanistic cohort. Prepared rather than raw semen was used for tests of DNA integrity.ConclusionsPICSI offered no clear advantage in relation to the primary outcome. PICSI led to a reduced miscarriage risk, but had no effect on CPR or preterm LBR.Future workRe-evaluate PICSI focusing on CP and miscarriage rates and consider aspects of sperm quality that PICSI favours.Trial registrationCurrent Controlled Trials ISRCTN99214271.FundingThis project was funded by the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership. The research is also supported by the NIHR Infrastructure at Leeds and the NIHR Clinical Research Network.

KIAA1199 is a secreted molecule that enhances osteoblastic stem cell migration and recruitment

Factors mediating mobilization of osteoblastic stem and progenitor cells from their bone marrow niche to be recruited to bone formation sites during bone remodeling are poorly known. We have studied secreted factors present in the bone marrow microenvironment and identified KIAA1199 (also known as CEMIP, cell migration inducing hyaluronan binding protein) in human bone biopsies as highly expressed in osteoprogenitor reversal cells (Rv.C) recruited to the eroded surfaces (ES), which are the future bone formation sites. In vitro, KIAA1199 did not affect the proliferation of human osteoblastic stem cells (also known as human bone marrow skeletal or stromal stem cells, hMSCs); but it enhanced cell migration as determined by scratch assay and trans-well migration assay. KIAA1199 deficient hMSCs (KIAA1199down) exhibited significant changes in cell size, cell length, ratio of cell width to length and cell roundness, together with reduction of polymerization actin (F-actin) and changes in phos-CFL1 (cofflin1), phos-LIMK1 (LIM domain kinase 1) and DSTN (destrin), key factors regulating actin cytoskeletal dynamics and cell motility. Moreover, KIAA1199down hMSC exhibited impaired Wnt signaling in TCF-reporter assay and decreased expression of Wnt target genes and these effects were rescued by KIAA1199 treatment. Finally, KIAA1199 regulated the activation of P38 kinase and its associated changes in Wnt-signaling. Thus, KIAA1199 is a mobilizing factor that interacts with P38 and Wnt signaling, and induces changes in actin cytoskeleton, as a mechanism mediating recruitment of hMSC to bone formation sites.

Origin and diversification of the plasminogen activation system among chordates

BackgroundThe plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce.ResultsOur phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)—and three-LU domain containing genes in general—occurred later in evolution and was first detectable after coelacanths.ConclusionsThis study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members—hitherto unknown in mammals—provide new perspectives on the evolution of this important enzyme system.

Inhibitory effects of Sanguisorba officinalis root extract on HYBID (KIAA1199)‐mediated hyaluronan degradation and skin wrinkling

Hyaluronan (HA), an important constituent of extracellular matrix in the skin, has many biological activities such as hydration that contributes to firmness and bounciness of the skin. We have reported that reduction in HA in the papillary dermis and over-expression of HYBID (HYaluronan Binding protein Involved in hyaluronan Depolymerization, alias KIAA1199 or CEMIP), a key molecule for HA degradation in skin fibroblasts, are implicated in facial skin wrinkling in Japanese and Caucasian women. However, little or no information is available for substances which inhibit the HYBID-mediated HA degradation. Inhibition of Sanguisorba officinalis root extract and ziyuglycoside I, one of the components of Sanguisorba officinalis root extract, to the HYBID-mediated HA degradation was assessed by size-exclusion chromatography of HA depolymerized by stable transfectants of HYBID in HEK293 cells (HYBID/HEK293 cells) or normal human skin fibroblasts (Detroit 551 cells and NHDF-Ad cells). The HYBID mRNA and protein expression was examined by quantitative real-time PCR and immunoblotting in the skin fibroblasts treated with Sanguisorba officinalis root extract, and size distribution of newly produced HA was evaluated by preparing metabolically radiolabelled HA. A double-blind, randomized and placebo-controlled study was carried out in the 21 healthy Japanese women, who were topically treated with the formulation containing Sanguisorba officinalis root extract or the placebo on each side of the face including crow's foot area. Sanguisorba officinalis root extract, but not ziyuglycoside I, abolished HYBID-mediated HA degradation by HYBID/HEK293 cells. Sanguisorba officinalis root extract also inhibited HYBID-mediated HA degradation in skin fibroblasts by down-regulating HYBID mRNA and protein expression. Although control untreated skin fibroblasts produced polydispersed HA, the cells treated with Sanguisorba officinalis root extract produced only high-molecular-weight HA. Treatment with Sanguisorba officinalis root extract-formulated lotion significantly improved skin elasticity, and reduced skin wrinkling scores at the outer eye corner compared with the placebo formulation. Sanguisorba officinalis root extract showed an anti-HYBID-mediated HA degradation activity and anti-wrinkle activity on human facial skin, which is accompanied by the improvement in elasticity. Our study provides the possibility of a new strategy to inhibit HYBID-mediated HA degradation for anti-wrinkle care.

Comparison of conventional slow freeze versus permeable cryoprotectant-free vitrification of abnormal semen sample: A randomized controlled trial

Background:The cryopreservation of semen samples by slow freezing remains as standard protocol. Recently, vitrification of spermatozoa was successfully reported with superior outcome. Till date, there is no randomized trial comparing the two different protocols.Aim:The aim of the present study is to evaluate the slow freezing with vitrification of the subfertile men spermatozoa to evaluate the progressive motility, vitality, and chromatin integrity.Setting:The study was conducted at University teaching hospital.Design:Study design involves randomized control trial.Materials and Methods:Twenty subfertile men with semen characteristics of severe oligoasthenozoospermia (SOA) and very SOA (VSOA) randomized to undergo slow freezing and vitrification protocol and cryopreserved at 1-month and 6-month storage interval, postthawed or warmed, samples were assessed for progressive motility, vitality, and hyaluronan binding. SPSS version 14 software was used for statistical analysis.Results:The SOA samples at 1 month revealed significantly higher motility (42% [22%–74%] vs. 7% [1%–13%]; P = 0.015) and vitality (57% [45%–78%] vs. 34.5% [27–42]; P < 0.001) following vitrification compared to slow-freeze method. For Very severe oligoasthenozoospermia (VSOA), the motility was significantly higher following vitrification (14.5% [2%–32%] vs. 2.5% [0%–4%]; P = 0.007). At 6 months, no statistically significant difference in motility was found between the two groups for Severe Oligoasthenozoospermia (SOA) samples (27% [13%–62%] vs. 8% [0%–11%]; P = 0.066), but motility was significantly higher following vitrification for VSOA samples (12.5% [3%–32%] vs. 2% [1%–5%]; P = 0.019). The hyaluronan-binding assay was comparable in both the groups at 6 months.Conclusions:The current study found the vitrification method involving the use of only nonpermeable cryoprotectants for cryopreservation of abnormal semen sample to be an effective alternative to the conventional slow-freeze technique.

Hyaluronan and Its Interactions With Immune Cells in the Healthy and Inflamed Lung.

Hyaluronan is a hygroscopic glycosaminoglycan that contributes to both extracellular and pericellular matrices. While the production of hyaluronan is essential for mammalian development, less is known about its interaction and function with immune cells. Here we review what is known about hyaluronan in the lung and how it impacts immune cells, both at homeostasis and during lung inflammation and fibrosis. In the healthy lung, alveolar macrophages provide the first line of defense and play important roles in immunosurveillance and lipid surfactant homeostasis. Alveolar macrophages are surrounded by a coat of hyaluronan that is bound by CD44, a major hyaluronan receptor on immune cells, and this interaction contributes to their survival and the maintenance of normal alveolar macrophage numbers. Alveolar macrophages are conditioned by the alveolar environment to be immunosuppressive, and can phagocytose particulates without alerting an immune response. However, during acute lung infection or injury, an inflammatory immune response is triggered. Hyaluronan levels in the lung are rapidly increased and peak with maximum leukocyte infiltration, suggesting a role for hyaluronan in facilitating leukocyte access to the injury site. Hyaluronan can also be bound by hyaladherins (hyaluronan binding proteins), which create a provisional matrix to facilitate tissue repair. During the subsequent remodeling process hyaluronan concentrations decline and levels return to baseline as homeostasis is restored. In chronic lung diseases, the inflammatory and/or repair phases persist, leading to sustained high levels of hyaluronan, accumulation of associated immune cells and an inability to resolve the inflammatory response.

Hapln4/Bral2 is a selective regulator for formation and transmission of GABAergic synapses between Purkinje and deep cerebellar nuclei neurons.

Purkinje cells (PCs) convey the sole output of the cerebellar cortex to the deep cerebellar nuclei (DCN). DCN neurons are enwrapped in densely organized extracellular matrix structures, known as perineuronal nets (PNNs). PNNs are typically found around fast-spiking GABAergic interneurons expressing parvalbumin but interestingly also exist surrounding other neurons, such as the neurons in the DCN and medial nucleus of the trapezoid body, which are the post-synaptic neurons of large axo-somatic synapses adapted for fast signaling. This characteristic localization prompted the hypothesis that PNNs might play a role in the maintenance and formation of large fast-signaling synapses. To elucidate the role of the PNN at these synapses, we investigated the electrophysiological and morphological properties of DCN synapses in hyaluronan and proteoglycan binding link protein 4 (Hapln4/Bral2) knockout (KO) mice around postnatal day (P)14. Hapln4/Bral2 is important for PNN structure, as it stabilizes the interaction between hyaluronan and proteoglycan. Here, using immunohistochemistry we show that Hapln4/Bral2 localized closely with GABAergic terminals. In DCN neurons of Hapln4/Bral2 KO mice, inhibitory synaptic strengths were reduced as compared to those in wild-type mice, whereas the properties of excitatory synapses were unaffected. The reduced IPSC amplitudes were mainly because of reduced numbers of releasable vesicles. Moreover, Hapln4/Bral2 deficiency reduced the number of PC GABAergic terminals in the DCN. These results demonstrate that Hapln4/Bral2 is a PNN component that selectively contributes to formation and transmission of PC-DCN synapses in the cerebellum. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

A truncated RHAMM protein for discovering novel therapeutic peptides

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7 kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7 kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7 kDa RHAMM. Therefore, in terms of its key binding properties, the 7 kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.

Interaction of Insulin-Like Growth Factor-Binding Protein 3 With Hyaluronan and Its Regulation by Humanin and CD44.

Insulin-like growth factor-binding protein-3 (IGFBP-3) belongs to a family of IGF-binding proteins. Humanin is a peptide known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including hyaluronan (HA). Here, we characterized the binding affinities of the IGFBP-3 protein and peptide (215-KKGFYKKKQCRPSKGRKR-232) to HA and to humanin and found that HA binds with a weaker affinity to this region than does humanin. Either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously. The HA receptor, CD44, blocked HA binding to IGFBP-3 but had no effect on binding of humanin to either IGFBP-3 or its peptide. Upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA. We show that IGFBP-3 and humanin in the medium of A549 lung cancer cells can immunoprecipitate in a complex. However, the fraction of IGFBP-3 in the medium that is able to bind HA was not complexed with humanin suggesting that HA binding to the 215-232 segment renders it inaccessible for binding to humanin. Moreover, while the cytotoxic effects of IGFBP-3 on cell viability were reversed by humanin, blocking HA-CD44 interaction with an anti-CD44 antibody in combination with IGFBP-3 did not have an additive negative effect on cell viability suggesting that IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions.

Development of In Vitro Methodologies to Investigate Binding by Sodium Hyaluronate in Eye Drops to Corneal Surfaces.

Purpose:To develop in vitro methods to assess binding by sodium hyaluronate in eye drops to corneal surfaces.Methods:Two different, complementary corneal binding set-ups were developed. In a dynamic in vitro model, confluent corneal epithelial cells (HCE-T) were assembled in chamber slides and a declining channel. A static model was constructed with ex vivo porcine corneas clamped in Franz cells. To test the predictive capacity of models, four different eye drops containing sodium hyaluronate were spiked with tritium-labeled sodium hyaluronate to standardize quantification. In both settings, eye drops were applied for 5 min and physiological conditions were mimicked by flushing with artificial tear fluid. Spreading experiments on HCE-T next to synthetic membranes were used for further characterization.Results:Binding was more pronounced in dynamic HCE-T model. Three of the four eye drops demonstrated sigmoidal elution of sodium hyaluronate, suggesting pronounced binding. One solution eluted distinctly faster, likewise the buffer control. The static method produced a similar ranking but at lower levels. When eye drops in which phosphate buffer was replaced by citrate buffer (i.e., to prevent calcification) were used, binding was not influenced. All eye drops spread immediately when placed on HCE-T and at the same order of magnitude on glass and polyethylene terephthalate surfaces.Conclusion:Dynamic and static models performed on different corneal sources were used to determine sodium hyaluronate binding kinetics in solutions under physiological conditions. These methodologies resulted in a ranking of the capacity of sodium hyaluronate to bind in vitro to corneal surfaces.

Targeted deletion of HYBID (hyaluronan binding protein involved in hyaluronan depolymerization/ KIAA1199/CEMIP) decreases dendritic spine density in the dentate gyrus through hyaluronan accumulation

HYBID (hyaluronan binding protein involved in hyaluronan [HA] depolymerization, KIAA1199/CEMIP) is a key player in HA depolymerization of the skin fibroblasts, arthritic synovial fibroblasts, and brain. Our previous study demonstrated that Hybid knock-out (KO) mice showed spatial memorial impairment, which is accompanied by the accumulation of high molecular weight HA in the hippocampus. However, the mechanism underlying cognitive impairment by Hybid deficiency remains unclear. In the present study, we examined the HA distribution patterns in the brains of wild-type (WT) and Hybid KO mice by HA staining using HA binding protein, and found that in Hybid KO mice, HA is accumulated and doublecortin-positive immature neurons are significantly decreased in the dentate gyrus of the hippocampus, where Hybid mRNA is highly expressed in WT mice. The Golgi-Cox staining demonstrated that the dendritic spine density is significantly decreased in the dentate gyrus granule cells in Hybid KO mice. These results suggest that Hybid-mediated HA degradation is critical for the synaptic formation process by contributing to cognitive functions, such as learning and memory, in the mouse brain.

Abstract 3742: Vitamin D inhibits hyaluronan synthesis and extracellular matrix gene expression in triple negative breast cancer

Abstract Human breast tumors with abundant hyaluronan (HA) and high expression of the HA synthesizing enzyme HAS2 are aggressive and exhibit poorer survival than tumors with low HA/HAS2. The vitamin D receptor (VDR) and its ligand 1,25(OH)2D3 (1,25D) exert anti-tumor effects in cancer cells and inhibit breast cancer growth in animals, but considerable heterogeneity has been reported in different model systems. Genomic profiling has demonstrated that, while a few genes are commonly regulated by 1,25D in all VDR positive cell lines, the majority of VDR regulated genes are cell line specific. In general, cell lines derived from the more aggressive tumor subtypes such as Triple Negative Breast Cancer (TNBC) express low levels of VDR and are less sensitive to 1,25D than cell lines derived from less aggressive subtypes. Through genomic profiling of TNBC cells derived from wild-type (WT) and VDR knockout (VDRKO) mice, we identified HAS2 as a 1,25D repressed gene. To understand the mechanistic impact of vitamin D and Has2 on HA synthesis and tumorigenic potential in TNBC cells, we utilized the human TNBC cell line Hs578T, which is representative of the mesenchymal/stem-like (MSL) subtype of TNBC. 1,25D inhibited HAS2 expression and HA synthesis in Hs578T cells, and also down-regulated CD44, a breast cancer stem cell marker which acts as the receptor for HA, the product of HAS2. Further studies determined that Hs578T cells are heterogeneous with respect to morphology and HAS2 expression. We therefore sorted parental Hs578T cells into HAhigh and HAlow populations. HAhigh populations exhibited elevated HA production, smaller size, increased proliferation and higher motility than HAlow populations. Through genomic profiling, HAhigh populations demonstrated higher levels of expression of genes involved in the enzymatic production and degradation of HA (HAS2, HAS2-AS, HYAL1), expression of extracellular matrix (ECM) proteins involved in HA binding and motility (PTX3, CEMIP, ACAN) and epithelial-mesenchymal transition (SNAI1) compared to HAlow populations. Despite their more aggressive phenotype, HAhigh populations retained expression of VDR protein at levels comparable to that of parental Hs578T cells and HAlow subclones. Treatment with 1,25D decreased production of HA in both HAhigh and HAlow populations, but significantly reduced expression of genes involved in the production of HA (HAS2, HAS2-AS) and HA binding and motility (ACAN, CEMIP) only in the HAhigh populations. This study suggests that 1,25D/VDR can suppress HAS2 and HA production and signaling in the ECM, and thus may reduce the aggressive phenotype of TNBC. Citation Format: Carmen J. Narvaez, Seamus Balinth, JoEllen Welsh. Vitamin D inhibits hyaluronan synthesis and extracellular matrix gene expression in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3742.

Abstract 2101: The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44

Abstract The abnormal accumulation of hyaluronan in the solid tumor microenvironment is associated with poor prognosis in several human cancers. Hyaluronan often accumulates in the tumor stroma. To understand the role of hyaluronan-accumulating stroma in tumor progression, we engineered a hyaluronan-producing fibroblast cell line 3T3HAS3 by lentiviral transduction of the human hyaluronan synthase 3 gene into Balb/c 3T3 cells. The 3T3HAS3 cells produced significant amounts of hyaluronan in vitro, which bind to the cell surface of the human breast cancer cell line MDA-MB-468. When co-grafted with MDA-MB-468 cells in nude mice, 3T3HAS3 significantly enhanced tumor growth. Immunohistochemical analysis of tumor xenografts showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of this co-graft model required hyaluronan production from 3T3HAS3 cells, as demonstrated by the delayed tumor growth upon hyaluronan removal by expression of the human PH20 gene in 3T3HAS3 cells, or by systemic administration of PEGylated recombinant human PH20 (PEGPH20). In contrast, neither the expression of CD44, a well-characterized hyaluronan receptor, in tumor and stromal fibroblasts, nor the binding of hyaluronan to CD44 in MDA-MB-468 tumor cells was essential for tumor growth. Small scale screening of signaling changes in xenograft tumors suggested that the AMPK/mTOR pathway may respond to hyaluronan removal by PEGPH20. Collectively, these data demonstrate that the growth of an engineered breast cancer xenograft model with hyaluronan-accumulating stroma is dependent on hyaluronan, and that hyaluronan-CD44 interaction may not be the main mechanism through which hyaluronan promotes tumor progression in certain tumors. Citation Format: Chunmei Zhao, Benjamin J. Thompson, Kelly Chen, Mathieu Marella, Susan Zimmerman, Trevor Kimbler, Barbara Blouw, Sheryl Garrovillo, Lei Huang, Adrian Radi, Zhongdong Huang, H. Michael Shepard, Sanna Rosengren, Christopher D. Thanos, Daniel C. Maneval. The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2101.

Investigating the Glycating Effects of Glucose, Glyoxal and Methylglyoxal on Human Sperm

Glycation is the non-enzymatic reaction between reducing sugars, such as glucose, and proteins, lipids or nucleic acids, producing Advanced Glycation End (AGE) products. AGEs, produced during natural senescence as well as through lifestyle factors such as diet and smoking, are key pathogenic compounds in the initiation and progression of diabetes. Importantly, many of these factors and conditions also have influence on male fertility, affecting sperm count and semen quality, contributing to the decreasing trend in male fertility. This study investigated the impact of AGEs on sperm damage. In vitro sperm glycation assays were used to determine the levels and localization of the potent AGE compound, carboxymethyl-lysine (CML) in response to treatment with the glycating compounds glucose, glyoxal and methylglyoxal. Sperm function assays were then used to assess the effects of glycation on motility and hyaluronan binding, and levels of oxidative DNA damage were analyzed through measurement of the marker, 8-oxoguanine. Results showed that glyoxal, but not glucose or methylglyoxal, induced significant increases in CML levels on sperm and this correlated with an increase in 8-oxoguanine. Immunocytochemistry revealed that AGEs were located on all parts of the sperm cell and most prominently on the head region. Sperm motility and hyaluronidase activity were not adversely affected by glycation. Together, the observed detrimental effects of the increased levels of AGE on DNA integrity, without an effect on motility and hyaluronidase activity, suggest that sperm may retain some fertilizing capacity under these adverse conditions.

Reduction of hyaluronan and increased expression of HYBID (alias CEMIP and KIAA1199) correlate with clinical symptoms in photoaged skin

Hyaluronan (HA) metabolism in skin fibroblasts is mediated by HYBID (hyaluronan binding protein involved in hyaluronan depolymerization, alias CEMIP and KIAA1199) and the HA synthases HAS1 and HAS2. However, photoageing-dependent changes in HA and their molecular mechanisms, and the relationship between HA metabolism and clinical symptoms in photoaged skin remain elusive. We examined the amount, size and tissue distribution of HA and expression levels of HYBID, HAS1 and HAS2 in photoaged skin, and analysed their relationship with the degree of photoageing. Photoageing-dependent changes of HA were investigated by studying skin biopsies isolated from photoprotected and photoexposed areas of the same donors, and the relationships between HA and photoageing symptoms such as skin wrinkling and sagging were examined. Skin biopsy specimens showed that the amount and size of HA are decreased in photoexposed skin compared with photoprotected skin, and this was accompanied by increased expression of HYBID and decreased expression of HAS1 and HAS2. Histologically, HA staining in the papillary dermis was decreased in photoexposed skin, showing reverse correlation with HYBID expression. HYBID expression in the photoexposed skin directly correlated with skin roughness and sagging parameters, and the reduced HA staining in the papillary dermis in the photoexposed skin positively correlated with these symptoms. These data demonstrate that imbalance between HYBID-mediated HA degradation and HAS-mediated HA synthesis may contribute to enhanced HA catabolism in photoaged skin, and suggest that HYBID-mediated HA reduction in the papillary dermis is related to skin wrinkling and sagging of photoaged skin.

CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment.

CD44 is a widely expressed cell adhesion molecule that binds to the extracellular matrix component, hyaluronan. However, this interaction is not constitutive in most immune cells at steady state, as the ability of CD44 to engage hyaluronan is highly regulated. While activated T cells and macrophages gain the ability to bind hyaluronan by CD44, the status in other immune cells is less studied. Here we found a percentage of murine eosinophils, natural killer and natural killer T cells were capable of interacting with hyaluronan at steady state. To further investigate the consequences of hyaluronan binding by CD44 in the hematopoietic system, point mutations of CD44 that either cannot bind hyaluronan (LOF-CD44) or have an increased affinity for hyaluronan (GOF-CD44) were expressed in CD44-deficient bone marrow. Competitive bone marrow reconstitution of irradiated mice revealed an early preference for GOF-CD44 over WT-CD44 expressing cells, and for WT-CD44 over LOF-CD44 expressing cells, in the hematopoietic progenitor cell compartment. The advantage of the hyaluronan-binding cells was observed in the hematopoietic stem and progenitor populations, and was maintained throughout the immune system. Hematopoietic stem cells bound minimal hyaluronan at steady state, and this was increased when the cells were induced to proliferate whereas multipotent progenitors had an increased ability to bind hyaluronan at steady state. In vitro, the addition of hyaluronan promoted their proliferation. Thus, proliferating hematopoietic progenitors bind hyaluronan, and hyaluronan binding cells have a striking competitive advantage in bone marrow engraftment.

Microenvironment-induced CD44v6 promotes early disease progression in chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia (CLL) outgrowth depends on signals from the microenvironment. We have previously found that in vitro reconstitution of this microenvironment induces specific variant isoforms of the adhesion molecule CD44, which confer human CLL with high affinity to hyaluronan (HA). Here, we determined the in vivo contribution of standard CD44 and its variants to leukemic B-cell homing and proliferation in Tcl1 transgenic mice with a B-cell–specific CD44 deficiency. In these mice, leukemia onset was delayed and leukemic infiltration of spleen, liver, and lungs, but not of bone marrow, was decreased. Competitive transplantation revealed that CLL homing to spleen and bone marrow required functional CD44. Notably, enrichment of CD44v6 variants particularly in spleen enhanced CLL engraftment and proliferation, along with increased HA binding. We recapitulated CD44v6 induction in the human disease and revealed the involvement of MAPK and NF-κB signaling upon CD40 ligand and B-cell receptor stimulation by in vitro inhibition experiments and chromatin immunoprecipitation assays. The investigation of downstream signaling after CD44v6-HA engagement uncovered the activation of extracellular signal-regulated kinase and p65. Consequently, anti-CD44v6 treatment reduced leukemic cell proliferation in vitro in human and mouse, confirming the general nature of the findings. In summary, we propose a CD44-NF-κB-CD44v6 circuit in CLL, allowing tumor cells to gain HA binding capacity and supporting their proliferation.

Hyaluronan-binding by CD44 reduces the memory potential of activated murine CD8 T cells.

Expansion and death of effector CD8 T cells are regulated to limit immunopathology and cells that escape contraction go on to generate immunological memory. CD44, a receptor for the extracellular matrix component hyaluronan, is a marker of activated and memory T cells. Here, we show with a murine model that the increase in CD44 expression and hyaluronan binding induced upon CD8 T cell activation was proportional to the strength of TCR engagement, thereby identifying the most strongly activated T cells. When CD44-/- and CD44+/+ OT-I CD8 T cells were adoptively transferred into mice challenged with Listeria-OVA, there was a slight increase in the percentage of CD44+/+ cells at the effector site. However, CD44+/+ cells were out-competed by CD44-/- cells after the contraction phase in the lymphoid tissues, and the CD44-/- cells preferentially formed more memory cells. The hyaluronan-binding CD44+/+ CD8 effector T cells showed increased pAkt expression, higher glucose uptake, and were more susceptible to cell death during the contraction phase compared to non-binding CD44+/+ and CD44-/- OT-I CD8 T cells, suggesting that CD44 and its engagement with hyaluronan skews CD8 T cells toward a terminal effector differentiation state that reduces their ability to form memory cells.