bims-supasi 2022-06-19 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2022‒06‒19
fourteen papers selected by
Jonathan Wolf Mueller
University of Birmingham

Adherence of Candida albicans and Malassezia Species to Skin Cells Induces Changes in the Expression of Genes Responsible for Heparan and Chondroitin Sulfate Chain Synthesis.
Experimental and Computational Study of Hyaluronidase Interactions with Glycosaminoglycans and their Ligands.
The "Loss" of Perineuronal Nets in Alzheimer's Disease: Missing or Hiding in Plain Sight?
Heparan sulfate dependent binding of plasmatic von Willebrand factor to blood circulating melanoma cells attenuates metastasis.
Chitosan sulfate-lysozyme hybrid hydrogels as platforms with fine-tuned degradability and sustained inherent antibiotic and antioxidant activities.
Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.
Increased serum concentrations of estrogen-induced growth factors Midkine and FGF2 in NF1 patients with plexiform neurofibroma.
Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota.
Proteoglycan Sulphation in the Function of the Mature Central Nervous System.
Identification of Heparan Sulfate in Dilated Cardiomyopathy by Integrated Bioinformatics Analysis.
Chondroitin Sulfates Control Invasiveness of the Basal-Like Breast Cancer Cell Line MDA-MB-231 Through ROR1.
Differential proteomics analysis of JEG-3 and JAR placental cell models and the effect of androgen treatment.
Pharmacokinetic, Hemostatic, and Anticancer Properties of a Low-Anticoagulant Bovine Heparin.
The enhancing effects of heparin on the biological activity of FGF-2 in heparin-FGF-2-calcium phosphate composite layers.

Actas Dermosifiliogr. 2022 Jun 09. pii: S0001-7310(22)00478-1. [Epub ahead of print]

Adherence of Candida albicans and Malassezia Species to Skin Cells Induces Changes in the Expression of Genes Responsible for Heparan and Chondroitin Sulfate Chain Synthesis.

H Ordiales, F Vázquez-López, M Pevida, B Vázquez-Losada, F Vázquez, L M Quirós, C Martín.

  Superficial fungal infections are common in dermatology and are often caused by opportunistic species in the Candida and Malassezia genera. The aim of this study was to analyze changes in the expression of genes coding for enzymes involved in the biosynthesis of glycosaminoglycan (GAG) chains following the adherence of Candida and Malassezia yeasts to skin cell lines. Gene expression was analyzed using reverse transcriptase-quantitative polymerase chain reaction assays. Interactions between the yeasts and the skin cells induced the following changes in genes involved in the biosynthesis of heparan sulfate and chondroitin sulfate: downregulation of CHPF in keratinocytes and downregulation of EXT1, EXT2, CHSY3, and CHPF in fibroblasts. Adherence to fibroblasts had an even greater effect on GAG biosynthetic enzymes, inducing the downregulation of 13 genes and the upregulation of 2 (CHST15 and CHST7). Interactions between yeasts and skin cells might affect the binding affinity of GAG chains, possibly changing their ability to function as receptors for pathogens and interfering with a key stage at the start of infection.

Keywords:  Chondroitin sulfates; Glycosaminoglycans; Infections; Reverse transcriptase polymerase chain reaction; condroitín sulfato; glicosaminoglicanos; heparan sulfate; heparán sulfato; infección; qRT-PCR

DOI:  https://doi.org/10.1016/j.ad.2022.06.004
Curr Mol Med. 2022 ;22(8): 675-690

Experimental and Computational Study of Hyaluronidase Interactions with Glycosaminoglycans and their Ligands.

Alexander V Maksimenko, Yuliya S Sakharova, Robert S Beabealashvilli.

  Covalent conjugation of hyaluronidase with copolymeric glycosaminoglycans (GAG, heparin and dermatan sulfate) considerably inactivates the enzyme, while conjugation with polymeric GAG (chondroitin sulfate and hyaluronan) improves its stability. These effects are associated with structural differences of these GAG caused by С-5 epimerization of glucuronic and iduronic acid residues and different effects of (α[1 - 4] and α[1 - 3] relative to β[1 - 4] and β[1 - 3]) glycosidic bonds. Pronounced effects of galactose C-4 epimers (in comparison with glucose) and disaccharide mixture (lactose, cellobiose, maltose) on endoglycosidase activity of hyaluronidase emphasize the importance of its diversified multi-contact microenvironment. For a better understanding of the mechanisms regulating hyaluronidase activity, molecular docking and molecular dynamics were chosen. Stabilization effect of chondroitin ligands on heat inactivation of hyaluronidase was demonstrated. An increase in denaturation temperature by 10-15oC hampers blocking of the active site entrance and prevents the enzyme inactivation. Enzyme-GAG interactions were examined by molecular docking with molecular dynamic elaboration. Gradual chemical modification of hyaluronidase was based on the calculated sequence of preferential binding of GAG. Theoretically, covalent binding of chondroitin sulfate trimers at cs7 or cs7, cs1 and cs5 on the enzyme surface provides complete protection against heparin inhibition. Computational investigation of hyaluronidase microenvironment and interactions which limit the enzyme activity allows identification of the best GAG regulators of hyaluronidase endoglycosidase activity and their experimental verification.

Keywords:  3D enzyme structure; Hyaluronidase; glycosaminoglycans; ligands; molecular docking; molecular dynamics; mono- and disaccharides

DOI:  https://doi.org/10.2174/1566524021666211018113204
Front Integr Neurosci. 2022 ;16 896400

The "Loss" of Perineuronal Nets in Alzheimer's Disease: Missing or Hiding in Plain Sight?

Jarrad M Scarlett, Shannon J Hu, Kimberly M Alonge.

  Perineuronal nets (PNNs) are chondroitin-sulfate glycosaminoglycan (CS-GAG) containing extracellular matrix structures that assemble around neurons involved in learning, memory, and cognition. Owing to the unique patterning of negative charges stemming from sulfate modifications to the attached CS-GAGs, these matrices play key roles in mediating glycan-protein binding, signaling interactions, and charged ion buffering of the underlying circuitry. Histochemical loss of PNN matrices has been reported for a range of neurocognitive and neurodegenerative diseases, implying that PNNs might be a key player in the pathogenesis of neurological disorders. In this hypothesis and theory article, we begin by highlighting PNN changes observed in human postmortem brain tissue associated with Alzheimer's disease (AD) and corresponding changes reported in rodent models of AD neuropathology. We then discuss the technical limitations surrounding traditional methods for PNN analyses and propose alternative explanations to these historical findings. Lastly, we embark on a global re-evaluation of the interpretations for PNN changes across brain regions, across species, and in relation to other neurocognitive disorders.

Keywords:  Alzheimer's disease; chondroitin sulfates; extracellular matrix; glycosaminoglycans; immunohistochemistry; mass spectrometry; perineuronal nets; proteoglycans

DOI:  https://doi.org/10.3389/fnint.2022.896400
Matrix Biol. 2022 Jun 08. pii: S0945-053X(22)00082-8. [Epub ahead of print]

Heparan sulfate dependent binding of plasmatic von Willebrand factor to blood circulating melanoma cells attenuates metastasis.

Yuanyuan Wang, Xiaobo Liu, Tobias Obser, Alexander T Bauer, Martin Heyes, Sarah Starzonek, Mina Zulal, Karena Opitz, Leonie Ott, Sabine Riethdorf, Tobias Lange, Klaus Pantel, Gerd Bendas, Stefan W Schneider, Marion Kusche-Gullberg, Christian Gorzelanny.

  Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.

Keywords:  ADAMTS13: A disintegrin‐like and metalloproteinase with thrombospondin type 1 motif 13; AFM: Atomic force microscopy; CHPF: Chondroitin sulfate synthase 2; CHSY: Chondroitin sulfate synthase; CS: Chondroitin sulfate; CTCs: Circulating tumour cells; ECIS: Electric cell-impedance sensing; EXT1: Exostosin 1; EXT2: Exostosin 2; HA: Hyaluronic acid; HAS: Hyaluronan Synthase; HPSE: Heparanase; HS: Heparan sulfate; HUVECs: Human umbilical vein endothelial cells; HYAL: Hyaluronidase; Heparan sulfate, melanoma, von Willebrand factor, circulating tumor cells, metastasis; NDST: Glucosaminyl N-deacetylase/N-sulfotransferase; RICM: Reflection interference contrast microscopy; SAW: Surface acoustic wave; SDC: Syndecan; SMFS: Single molecular force spectroscopy; STED: Stimulated emission depletion; ULvWF: Ultra-large vWF; VCAM1: Vascular cell adhesion protein 1; VLA4: Very late antigen 4; WGA: Wheat germ agglutinin; vWF: von Willebrand factor

DOI:  https://doi.org/10.1016/j.matbio.2022.06.002
Carbohydr Polym. 2022 Sep 01. pii: S0144-8617(22)00516-1. [Epub ahead of print]291 119611

Chitosan sulfate-lysozyme hybrid hydrogels as platforms with fine-tuned degradability and sustained inherent antibiotic and antioxidant activities.

Antonio Aguanell, María Luisa Del Pozo, Carlos Pérez-Martín, Gabriela Pontes, Agatha Bastida, Alfonso Fernández-Mayoralas, Eduardo García-Junceda, Julia Revuelta.

  The control of the properties and biological activities of chitosan-lysozyme hybrid hydrogels to exploit their interesting biomedical applications depends largely on the chitosan acetylation pattern, a difficult parameter to control. Herein, we have prepared sulfated chitosan-lysozyme hydrogels as versatile platforms with fine-tuned degradability and persistent bactericidal and antioxidant properties. The use of chitosan sulfates instead of chitosan has the advantage that the rate and mechanisms of lysozyme release, as well as antibacterial and antioxidant activities, depend on the sulfation profile, a structural parameter that is easily controlled by simple chemical modifications. Thus, while 6-O-sulfated chitosan hydrogels allow the release of loaded lysozyme in a short time (60% in 24 h), due to a high rate of degradation that allows rapid antibiotic and antioxidant activities, in 3-O-sulfated systems there is a slow release of lysozyme (80% in 21 days), resulting in long-lasting antibiotic and antioxidant activities.

Keywords:  Antibiotic activity; Antioxidant activity; Chitosan sulfate; Lysozyme; Physicochemical parameters; Polymers

DOI:  https://doi.org/10.1016/j.carbpol.2022.119611
ACS Appl Mater Interfaces. 2022 Jun 16.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Charles-Francois V Latchoumane, Pradeep Chopra, Lifeng Sun, Aws Ahmed, Francesco Palmieri, Hsueh-Fu Wu, Rebecca Guerreso, Kristen Thorne, Nadja Zeltner, Geert-Jan Boons, Lohitash Karumbaiah.

  Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Keywords:  ERK; FGF2; click chemistry; neural stem cells; synthetic heparan sulfate hydrogel

DOI:  https://doi.org/10.1021/acsami.2c01575
Am J Transl Res. 2022 ;14(5): 3180-3188

Increased serum concentrations of estrogen-induced growth factors Midkine and FGF2 in NF1 patients with plexiform neurofibroma.

Isis Atallah, Ana M Cieza Rivera, Octavio M Rivero Lezcano, Laura Tascón-González, Carolina González-Cortés, Cristina Diez Tascón, Tania Fernández-Villa, Vicente Martín.

Neurofibromatosis type 1 (NF1) predisposes to the development of dermal and plexiform neurofibromas and serum of NF1 patients stimulates neurofibroma proliferation in vitro. This study aimed to determine whether, in NF1 patients, serum levels of midkine (MK) and fibroblast growth factor 2 (FGF2) were associated with the number and/or type of neurofibromas. In addition, their concentrations were correlated with serum levels of dehydroepiandrosterone sulfate (DHEAS), a neurosteroid secreted by the peripheral nervous system. We performed a case control-study and measured, by ELISA assay, serum concentrations of MK, FGF2, and DHEAS in 20 NF1 patients and 30 controls. We found increased serum levels of MK and FGF2 in NF1 patients between 30 and 50 years old. Their concentrations were significantly higher in NF1 patients with plexiform neurofibromas than in controls (P=0.003 for MK and P=0.008 for FGF2). As an underlying hormonal regulation was suspected, DHEAS serum levels were measured but no difference was observed between patients and controls. We also observed a strong association between MK and FGF2 levels (P=0.0001) in NF1 patients and controls. In conclusion, we point out MK and FGF2 as biomarkers for plexiform neurofibroma in NF1 patients. As both growth factors are estrogen-responsive genes and neurofibromin is a co-repressor of estrogen receptor alpha activity, we suggest that the increased serum levels of MK and FGF2 observed in NF1 patients might be due to estradiol hypersensitivity.

Keywords: ELISA; Neurofibromatosis type 1; dehydroepiandrosterone sulfate (DHEAS); fibroblast growth factor 2 (FGF2); midkine (MK); neurofibroma; plexiform
Nat Chem Biol. 2022 Jun 16.

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota.

Ana S Luis, Arnaud Baslé, Dominic P Byrne, Gareth S A Wright, James A London, Chunsheng Jin, Niclas G Karlsson, Gunnar C Hansson, Patrick A Eyers, Mirjam Czjzek, Tristan Barbeyron, Edwin A Yates, Eric C Martens, Alan Cartmell.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

DOI: https://doi.org/10.1038/s41589-022-01039-x
Front Integr Neurosci. 2022 ;16 895493

Proteoglycan Sulphation in the Function of the Mature Central Nervous System.

James W Fawcett, Jessica C F Kwok.

  Chondroitin sulphate and heparan sulphate proteoglycans (CSPGS and HSPGs) are found throughout the central nervous system (CNS). CSPGs are ubiquitous in the diffuse extracellular matrix (ECM) between cells and are a major component of perineuronal nets (PNNs), the condensed ECM present around some neurons. HSPGs are more associated with the surface of neurons and glia, with synapses and in the PNNs. Both CSPGs and HSPGs consist of a protein core to which are attached repeating disaccharide chains modified by sulphation at various positions. The sequence of sulphation gives the chains a unique structure and local charge density. These sulphation codes govern the binding properties and biological effects of the proteoglycans. CSPGs are sulphated along their length, the main forms being 6- and 4-sulphated. In general, the chondroitin 4-sulphates are inhibitory to cell attachment and migration, while chondroitin 6-sulphates are more permissive. HSPGs tend to be sulphated in isolated motifs with un-sulphated regions in between. The sulphation patterns of HS motifs and of CS glycan chains govern their binding to the PTPsigma receptor and binding of many effector molecules to the proteoglycans, such as growth factors, morphogens, and molecules involved in neurodegenerative disease. Sulphation patterns change as a result of injury, inflammation and ageing. For CSPGs, attention has focussed on PNNs and their role in the control of plasticity and memory, and on the soluble CSPGs upregulated in glial scar tissue that can inhibit axon regeneration. HSPGs have key roles in development, regulating cell migration and axon growth. In the adult CNS, they have been associated with tau aggregation and amyloid-beta processing, synaptogenesis, growth factor signalling and as a component of the stem cell niche. These functions of CSPGs and HSPGs are strongly influenced by the pattern of sulphation of the glycan chains, the sulphation code. This review focuses on these sulphation patterns and their effects on the function of the mature CNS.

Keywords:  chondroitin sulphate; heparan sulphate; memory; neurodegeneration; neuroregeneration; perineuronal net; plasticity; stem cells

DOI:  https://doi.org/10.3389/fnint.2022.895493
Front Cardiovasc Med. 2022 ;9 900428

Identification of Heparan Sulfate in Dilated Cardiomyopathy by Integrated Bioinformatics Analysis.

Wenyu Song, Fujian Lu, Zequan Ding, Liqi Huang, Kui Hu, Jinmiao Chen, Lai Wei.

  Objectives: Heparan sulfate (HS) forms heparan sulfate proteoglycans (HSPGs), such as syndecans (SDCs) and glypicans (GPCs), to perform biological processes in the mammals. This study aimed to explore the role of HS in dilated cardiomyopathy (DCM).Methods: Two high throughput RNA sequencing, two microarrays, and one single-cell RNA sequencing dataset of DCM hearts were downloaded from the Gene Expression Omnibus (GEO) database and integrated for bioinformatics analyses. Differential analysis, pathway enrichment, immunocytes infiltration, subtype identification, and single-cell RNA sequencing analysis were used in this study.
Results: The expression level of most HSPGs was significantly upregulated in DCM and was closely associated with immune activation, cardiac fibrosis, and heart failure. Syndecan2 (SDC2) was highly associated with collagen I and collagen III in cardiac fibroblasts of DCM hearts. HS biosynthetic pathway was activated, while the only enzyme to hydrolyze HS was downregulated. Based on the expression of HSPGs, patients with DCM were classified into three molecular subtypes, i.e., C1, C2, and C3. Cardiac fibrosis and heart failure were more severe in the C1 subtype.
Conclusion: Heparan sulfate is closely associated with immune activation, cardiac fibrosis, and heart failure in DCM. A novel molecular classification of patients with DCM is established based on HSPGs.

Keywords:  bioinformatics; cardiac inflammation and fibrosis; dilated cardiomyopathy; heparan sulfate; molecular subtype

DOI:  https://doi.org/10.3389/fcvm.2022.900428
Front Oncol. 2022 ;12 914838

Chondroitin Sulfates Control Invasiveness of the Basal-Like Breast Cancer Cell Line MDA-MB-231 Through ROR1.

Satomi Nadanaka, Jun-Ichi Tamura, Hiroshi Kitagawa.

  Extracellular and cell surface chondroitin sulfates (CSs) regulate cancer cell properties, including proliferation and invasion. Thus, it is necessary to understand the mechanisms underlying their roles in cancer. Although we have shown that CS has an inherent ability to enhance the invasive activity of the human triple-negative breast cancer cell line MDA-MB-231, its molecular mechanism remains elusive. Here, we focused on receptor tyrosine kinase-like orphan receptor 1 (ROR1) and dickkopf WNT signaling pathway inhibitor 1 (DKK1). MDA-MB-231 cells express high levels of ROR1; their invasive potential depends on ROR1 signaling. Although accumulating evidence has demonstrated that ROR1 is associated with aggressive breast-cancer phenotypes, the whole picture of its biological function remains poorly understood. In this study, we examined whether CS controls ROR1 function. Surface plasmon resonance analysis indicated that CSs were bound to ROR1 in the presence of WNT5A. The invasive activity of MDA-MB-231 cells enhanced by CSs was completely suppressed by ROR1 knockdown. In addition, knockdown of the CS biosynthetic enzymes CHST11 and CHST15 inhibited invasive activity, even in the presence of ROR1. These results suggest that CS is required to induce an ROR1-dependent, aggressive MDA-MB-231 phenotype. ROR1 signaling in MDA-MB-231 cells activated c-Jun N-terminal kinase (JNK), leading to increased invasive potential; moreover, exogenous CSs activated JNK. MDA-MB-231 cells express DKK1, a tumor suppressor factor that binds to CS, at high levels. Knockdown of DKK1 enhanced CS-stimulated tumor invasion activity of MDA-MB-231 cells, suggesting that DKK1 sequesters CS to block ROR1/JNK signaling. These results showed that CSs promotes cancer aggressiveness through the ROR1-JNK axis in MDA-MB-231 cells.

Keywords:  Dickkopf 1; breast cancer; chondroitin sulfate; proteoglycan; receptor tyrosine kinase-like orphan receptor 1

DOI:  https://doi.org/10.3389/fonc.2022.914838
J Steroid Biochem Mol Biol. 2022 Jun 08. pii: S0960-0760(22)00089-9. [Epub ahead of print] 106138

Differential proteomics analysis of JEG-3 and JAR placental cell models and the effect of androgen treatment.

Laken Kruger, Guihua Yue, Vijaya Saradhi Mettu, Alison Paquette, Sheela Sathyanarayana, Bhagwat Prasad.

  The placenta is a vital fetal organ that plays an important role in maintaining fetal sex hormone homeostasis. Xenobiotics can alter placental sex-steroidogenic enzymes and transporters, including enzymes such as aromatase (CYP19A1) and the hydroxysteroid dehydrogenases (HSDs) but studying how compounds disrupt in vivo placental metabolism is complex. Utilizing high-throughput in vitro models is critical to predict the disruption of placental sex-steroidogenic enzymes and transporters, particularly by drug candidates in the early stages of drug discovery. JAR and JEG-3 cells are the most common, simple, and cost-effective placental cell models that are capable of high-throughput screening, but how well they express the sex-steroidogenic enzymes and transporters is not well known. Here, we compared the proteomes of JAR and JEG-3 cells in the presence and absence of physiologically relevant concentrations of dehydroepiandrosterone (DHEA, 8µM) and testosterone (15nM) to aid the characterization of sex-steroidogenic enzymes and transporters in these cell models. Global proteomics analysis detected 2,931 and 3,449 proteins in JAR cells and JEG-3 cells, respectively. However, dramatic differences in sex-steroidogenic enzymes and transporters were observed between these cells. In particular, the basal expression of steroid sulfatase (STS), HSD17B1, and HSD17B7 were unique to JEG-3 cells. JEG-3 cells also showed significantly higher protein levels of aldo-keto reductase (AKR) 1A1 and AKR1B1, while JAR cells showed significantly higher levels of HSD17B4 and HSDB12. Aldehyde dehydrogenase (ALDH) 3A2 and HSD17B11 enzymes as well as the transporters sterol O-acyltransferase (SOAT) 1 and ATP binding cassette subfamily G2 (ABCG2) were comparable between the cell lines, whereas sulfotransferases (SULTs) were uniquely present within JAR cells. Androgen treatments significantly lowered HSD17B11 HSD17B4, HSD17B12, and ALDH3A2 levels in JAR cells. DHEA treatment significantly raised the level of HSD17B1 by 51% in JEG-3 cells, whereas CYP19A1 was increased to significant levels in both JAR and JEG-3 cells after androgen treatments. The proteomics data were supported by a complementary targeted metabolomics analysis of culture media in the DHEA (8µM) and testosterone (15nM) treated groups. This study has indicated that untreated JEG-3 cells express more sex-steroidogenic enzymes and transporters. Nevertheless, JEG-3 and JAR cells are unique and their respective proteomics data can be used to select the best model depending on the hypothesis.

Keywords:  Androgen metabolism; JAR; JEG-3; Placenta; Proteomics; and Steroids

DOI:  https://doi.org/10.1016/j.jsbmb.2022.106138
TH Open. 2022 Apr;6(2): e114-e123

Pharmacokinetic, Hemostatic, and Anticancer Properties of a Low-Anticoagulant Bovine Heparin.

Roberto P Santos, Ana M F Tovar, Marcos R Oliveira, Adriana A Piquet, Nina V Capillé, Stephan N M C G Oliveira, Ana H Correia, José N Farias, Eduardo Vilanova, Paulo A S Mourão.

  Heparin is a centennial anticoagulant drug broadly employed for treatment and prophylaxis of thromboembolic conditions. Although unfractionated heparin (UFH) has already been shown to have remarkable pharmacological potential for treating a variety of diseases unrelated with thromboembolism, including cancer, atherosclerosis, inflammation, and virus infections, its high anticoagulant potency makes the doses necessary to exert non-hemostatic effects unsafe due to an elevated bleeding risk. Our group recently developed a new low-anticoagulant bovine heparin (LABH) bearing the same disaccharide building blocks of the UFH gold standard sourced from porcine mucosa (HPI) but with anticoagulant potency approximately 85% lower (approximately 25 and 180 Heparin International Units [IU]/mg). In the present work, we investigated the pharmacokinetics profile, bleeding potential, and anticancer properties of LABH administered subcutaneous into mice. LABH showed pharmacokinetics profile similar to HPI but different from the low-molecular weight heparin (LMWH) enoxaparin and diminished bleeding potential, even at high doses. Subcutaneous treatment with LABH delays the early progression of Lewis lung carcinoma, improves survival, and brings beneficial health outcomes to the mice, without the advent of adverse effects (hemorrhage/mortality) seen in the animals treated with HPI. These results demonstrate that LABH is a promising candidate for prospecting new therapeutic uses for UFH.

Keywords:  Lewis lung carcinoma; bleeding; low-molecular weight heparin; pharmacokinetics; unfractionated heparin

DOI:  https://doi.org/10.1055/s-0042-1745743
Acta Biomater. 2022 Jun 10. pii: S1742-7061(22)00352-X. [Epub ahead of print]

The enhancing effects of heparin on the biological activity of FGF-2 in heparin-FGF-2-calcium phosphate composite layers.

Mayu Yasunaga, Fumiko Kobayashi, Yu Sogo, Kazutoshi Murotomi, Motohiro Hirose, Yuki Hara, Masashi Yamazaki, Atsuo Ito.

  Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the composite layers is important for its wider application in orthopedics and dentistry. This study incorporated low-molecular-weight heparin (LMWH) into the FGF-2-calcium phosphate composite layers and clarified the enhancing effects of LMWH on the biological activity of FGF-2 in the composite layers in vitro. LMWH-FGF-2-calcium phosphate composite layers were successfully formed on zirconia in supersaturated calcium phosphate solutions. The composite layers comprised continuous and macroscopically homogeneous layers and particles smaller than 500 nm in size composed of amorphous calcium phosphate. The amounts of Ca and P deposited on zirconia remained almost unchanged with the addition of LMWH under the presence of FGF-2 in the supersaturated calcium phosphate solution. The LMWH in the supersaturated calcium phosphate solution increased the stability of FGF-2 in the solution and the amount of FGF-2 in the composite layers. The LMWH in the composite layers increased the mitogenic and endothelial tube-forming activities of FGF-2, and FGF-2 activity of inducing osteogenic differentiation gene expression pattern in the composite layers. Our results indicate that the enhanced biological activity of FGF-2 in the LMWH-FGF-2-calcium phosphate composite layers is attributed to an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the supersaturated calcium phosphate solution and the composite layers. The LMWH-FGF-2-calcium phosphate composite layer is a promising coating for orthopedic and dental implants. STATEMENT OF SIGNIFICANCE: Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the layers is important for wider its application in orthopedics and dentistry. This study demonstrates the enhancing effects of low-molecular-weight heparin (LMWH) contained within LMWH-FGF-2-calcium phosphate composite layers on the biological activity of FGF-2 in vitro. Our results indicate that the enhanced biological activity of FGF-2 within the composite layers arises from an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the LMWH-FGF-2-calcium phosphate composite layers and supersaturated calcium phosphate solutions used for coating the composite layers.

Keywords:  Biological activity; Calcium phosphate coating; Fibroblast growth factor-2; Low-molecular-weight heparin; Orthopedic and dental implants

DOI:  https://doi.org/10.1016/j.actbio.2022.06.013