bims-supasi 2023-04-02 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2023‒04‒02
sixteen papers selected by
Jonathan Wolf Mueller
University of Birmingham

Heparin Mimetics and Their Impact on Extracellular Matrix Protein Assemblies.
Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest.
Dehydroepiandrosterone sulfate (DHEAS) in excreta is a good indicator of serum DHEAS in Japanese macaques (Macaca fuscata).
Analysis of Heparin Samples by Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy in the Solid State.
Exostosin 1 Knockdown Induces Chemoresistance in MV3 Melanoma Cells by Upregulating JNK and MEK/ERK Signaling.
Gut-Derived Uremic Toxins in CKD: An Improved Approach for the Evaluation of Serum Indoxyl Sulfate in Clinical Practice.
Structural Elucidation of a Glucan from Trichaster palmiferus by Its Degraded Products and Preparation of Its Sulfated Derivative as an Anticoagulant.
VALIDATION OF ENZYME-LINKED IMMUNOSORBENT ASSAY TECHNIQUES TO MEASURE SERUM DEHYDROEPIANDROSTERONE (DHEA) AND DHEA-S IN NARWHALS (MONODON MONOCEROS).
Tissue-specific expression of carbohydrate sulfotransferases drives keratan sulfate biosynthesis in the notochord and otic vesicles of Xenopus embryos.
Validation of Recombinant Heparan Sulphate Reagents for CNS Repair.
Heparan Sulfate and Enoxaparin Interact at the Interface of the Spike Protein of HCoV-229E but Not with HCoV-OC43.
Sulfate and acid-base balance.
DHEA and Its Metabolites Reduce the Cytokines Involved in the Inflammatory Response and Fibrosis in Primary Biliary Cholangitis.
Designing Smaller, Synthetic, Functional Mimetics of Sulfated Glycosaminoglycans as Allosteric Modulators of Coagulation Factors.
The Heparan Sulfate Proteoglycan Syndecan-1 Triggers Breast Cancer Cell-Induced Coagulability by Induced Expression of Tissue Factor.
Indoxyl-sulfate activation of the AhR- NF-κB pathway promotes interleukin-6 secretion and the subsequent osteogenic differentiation of human valvular interstitial cells from the aortic valve.

Pharmaceuticals (Basel). 2023 Mar 22. pii: 471. [Epub ahead of print]16(3):

Heparin Mimetics and Their Impact on Extracellular Matrix Protein Assemblies.

Fabian Heide, Manuel Koch, Jörg Stetefeld.

  Heparan sulfate is a crucial extracellular matrix component that organizes structural features and functional protein processes. This occurs through the formation of protein-heparan sulfate assemblies around cell surfaces, which allow for the deliberate local and temporal control of cellular signaling. As such, heparin-mimicking drugs can directly affect these processes by competing with naturally occurring heparan sulfate and heparin chains that then disturb protein assemblies and decrease regulatory capacities. The high number of heparan-sulfate-binding proteins that are present in the extracellular matrix can cause obscure pathological effects that should be considered and examined in more detail, especially when developing novel mimetics for clinical use. The objective of this article is to investigate recent studies that present heparan-sulfate-mediated protein assemblies and the impact of heparin mimetics on the assembly and function of these protein complexes.

Keywords:  clinical drug development; extracellular matrix organization; heparan-sulfate-binding proteins; heparin mimetics; protein aggregation; protein multimers; protein–ligand interactions

DOI:  https://doi.org/10.3390/ph16030471
J Am Chem Soc. 2023 Mar 31.

Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest.

Jerome Riedel, Maike Lettow, Márkó Grabarics, Michael Götze, Rebecca L Miller, Geert-Jan Boons, Gerard Meijer, Gert von Helden, Gergo Peter Szekeres, Kevin Pagel.

In recent years, glycosaminoglycans (GAGs) have emerged into the focus of biochemical and biomedical research due to their importance in a variety of physiological processes. These molecules show great diversity, which makes their analysis highly challenging. A promising tool for identifying the structural motifs and conformation of shorter GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this work, the cryogenic gas-phase IR spectra of mass-selected heparan sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to extract vibrational features that are characteristic to structural motifs. The data were augmented with chondroitin sulfate (CS) disaccharide spectra to assemble a training library for random forest (RF) classifiers. These were used to discriminate between GAG classes (CS or HS) and different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With optimized data preprocessing and RF modeling, a prediction accuracy of >97% was achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. These results exemplify the importance of combining gas-phase cryogenic IR ion spectroscopy with machine learning to improve the future analytical workflow for GAG sequencing and that of other biomolecules, such as metabolites.

DOI: https://doi.org/10.1021/jacs.2c12762
Gen Comp Endocrinol. 2023 Mar 23. pii: S0016-6480(23)00082-5. [Epub ahead of print]338 114277

Dehydroepiandrosterone sulfate (DHEAS) in excreta is a good indicator of serum DHEAS in Japanese macaques (Macaca fuscata).

Keiko Mouri, Keiko Shimizu.

  We developed a microplate enzyme immunoassay (EIA) to measure dehydroepiandrosterone sulfate (DHEAS) in the blood, urine, and feces of Japanese macaques and evaluated the relationship between serum DHEAS and excreta DHEAS. Our DHEAS EIA using heterological DHEA derivatives conjugated with enzyme was highly sensitive, and linearities and recoveries for all matrices of Japanese macaques were reliable. For the biological evaluation of the metabolism of DHEAS in Japanese macaques, dissolved DHEAS was injected into the subjects, and consecutively collected serum, urine, and fecal samples were analyzed. The peaks of serum DHEAS were observed 6 h after the administration, while those of urine and feces were observed after 24 h. The fluctuation of those in urine and feces were significantly correlated with serum DHEAS levels. In addition, we measured pregnanediol-glucuronide (PdG), and estrone-conjugate (E1C) in urine and fecal samples to investigate the effects of administrated DHEAS on these progesterone and estrogen metabolites. The peak of PdG was observed 24 h after administration, then declined sharply. The concentration of E1C increased 1 week after administration in two out of three subjects. Our results suggest that measuring urinary and fecal DHEAS with our EIA provides a non-invasive alternative to assessing DHEAS levels in the serum of Japanese macaques.

Keywords:  Adrenal hormone; Enzyme immunoassay; Non-human primate; Sex steroid

DOI:  https://doi.org/10.1016/j.ygcen.2023.114277
ACS Cent Sci. 2023 Mar 22. 9(3): 381-392

Analysis of Heparin Samples by Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy in the Solid State.

Anthony J Devlin, Courtney J Mycroft-West, Jeremy E Turnbull, Marcelo Andrade de Lima, Marco Guerrini, Edwin A Yates, Mark A Skidmore.

Heparin is a polydisperse, heterogeneous polysaccharide of the glycosaminoglycan (GAG) class that has found widespread clinical use as a potent anticoagulant and is classified as an essential medicine by the World Health Organization. The importance of rigorous monitoring and quality control of pharmaceutical heparin was highlighted in 2008, when the existing regulatory procedures failed to identify a life-threatening adulteration of pharmaceutical heparin with oversulfated chondroitin sulfate (OSCS). The subsequent contamination crisis resulted in the exploration of alternative approaches for which the use of multidimensional nuclear magnetic resonance (NMR) spectroscopy techniques and multivariate analysis emerged as the gold standard. This procedure is, however, technically demanding and requires access to expensive equipment. An alternative approach, utilizing attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) combined with multivariate analysis, has been developed. The method described enables the differentiation of diverse GAG samples, the classification of samples of distinct species provenance, and the detection of both established heparin contaminants and alien polysaccharides. This methodology has sensitivity comparable to that of NMR and can facilitate the rapid, cost-effective monitoring and analysis of pharmaceutical heparin. It is therefore suitable for future deployment throughout the supply chain.

DOI: https://doi.org/10.1021/acscentsci.2c01176
Int J Mol Sci. 2023 Mar 13. pii: 5452. [Epub ahead of print]24(6):

Exostosin 1 Knockdown Induces Chemoresistance in MV3 Melanoma Cells by Upregulating JNK and MEK/ERK Signaling.

Vladlena Pfeifer, Heiko Weber, Yuanyuan Wang, Martin Schlesinger, Christian Gorzelanny, Gerd Bendas.

  Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC50 values two- or fourfold, respectively. Resistance formation was scarcely related to HSPG deficiency, suggested by enzymatic cleavage of HSPG in control cells. Notably, EXT1kd induced an upregulation of EGFR signaling via JNK and MEK/ERK, and hence blocking these kinases returned resistance to a sensitive level. JNK appeared as a key signal component, also inducing higher migratory activity of EXT1kd cells. Furthermore, EXT1kd upregulated thrombotic properties of MV3 cells, indicated by tissue factor and PAR-1 expression, functionally reflected by a stronger activation of platelet aggregation. EXT1 was confirmed to act as a tumor suppressor, shown here for the first time to affect chemosensitivity of melanoma cells.

Keywords:  HSPG-heparan sulfate proteoglycan; cancer; chemoresistance; doxorubicin; exostosin1; melanoma; mitoxantrone

DOI:  https://doi.org/10.3390/ijms24065452
Int J Mol Sci. 2023 Mar 07. pii: 5142. [Epub ahead of print]24(6):

Gut-Derived Uremic Toxins in CKD: An Improved Approach for the Evaluation of Serum Indoxyl Sulfate in Clinical Practice.

Gianvito Caggiano, Loredana Amodio, Alessandra Stasi, Nicola Antonio Colabufo, Santina Colangiulo, Francesco Pesce, Loreto Gesualdo.

  In the past years, indoxyl sulfate has been strongly implicated in kidney disease progression and contributed to cardiovascular morbidity. Moreover, as a result of its elevated albumin affinity rate, indoxyl sulfate is not adequately cleared by extracorporeal therapies. Within this scenario, although LC-MS/MS represents the conventional approach for IS quantification, it requires dedicated equipment and expert skills and does not allow real-time analysis. In this pilot study, we implemented a fast and simple technology designed to determine serum indoxyl sulfate levels that can be integrated into clinical practice. Indoxyl sulfate was detected at the time of enrollment by Tandem MS from 25 HD patients and 20 healthy volunteers. Next, we used a derivatization reaction to transform the serum indoxyl sulfate into Indigo blue. Thanks to the spectral shift to blue, its quantity was measured by the colorimetric assay at a wavelength of 420-450 nm. The spectrophotometric analysis was able to discriminate the levels of IS between healthy subjects and HD patients corresponding to the LC-MS/MS. In addition, we found a strong linear relationship between indoxyl sulfate levels and Indigo levels between the two methods (Tandem MS and spectrophotometry). This innovative method in the assessment of gut-derived indoxyl sulfate could represent a valid tool for clinicians to monitor CKD progression and dialysis efficacy.

Keywords:  CKD; LC-MS/MS; hemodialysis; indoxyl sulfate; uremic toxins

DOI:  https://doi.org/10.3390/ijms24065142
Mar Drugs. 2023 Feb 24. pii: 148. [Epub ahead of print]21(3):

Structural Elucidation of a Glucan from Trichaster palmiferus by Its Degraded Products and Preparation of Its Sulfated Derivative as an Anticoagulant.

Haiqiong Ma, Qingxia Yuan, Hao Tang, Hongjie Tan, Tingting Li, Shiying Wei, Jinwen Huang, Yue Yao, Yaping Hu, Shengping Zhong, Yonghong Liu, Chenghai Gao, Longyan Zhao.

  Echinoderms have been attracting increasing attention for their polysaccharides, with unique chemical structure and enormous potential for preparing drugs to treat diseases. In this study, a glucan (TPG) was obtained from the brittle star Trichaster palmiferus. Its structure was elucidated by physicochemical analysis and by analyzing its low-molecular-weight products as degraded by mild acid hydrolysis. The TPG sulfate (TPGS) was prepared, and its anticoagulant activity was investigated for potential development of anticoagulants. Results showed that TPG consisted of a consecutive α1,4-linked D-glucopyranose (D-Glcp) backbone together with a α1,4-linked D-Glcp disaccharide side chain linked through C-1 to C-6 of the main chain. The TPGS was successfully prepared with a degree of sulfation of 1.57. Anticoagulant activity results showed that TPGS significantly prolonged activated partial thromboplastin time, thrombin time, and prothrombin time. Furthermore, TPGS obviously inhibited intrinsic tenase, with an EC50 value of 77.15 ng/mL, which was comparable with that of low-molecular-weight heparin (LMWH) (69.82 ng/mL). TPGS showed no AT-dependent anti-FIIa and anti-FXa activities. These results suggest that the sulfate group and sulfated disaccharide side chains play a crucial role in the anticoagulant activity of TPGS. These findings may provide some information for the development and utilization of brittle star resources.

Keywords:  anticoagulant activity; brittle star; glucan; polysaccharide; structure

DOI:  https://doi.org/10.3390/md21030148
J Zoo Wildl Med. 2023 Mar;54(1): 119-130

VALIDATION OF ENZYME-LINKED IMMUNOSORBENT ASSAY TECHNIQUES TO MEASURE SERUM DEHYDROEPIANDROSTERONE (DHEA) AND DHEA-S IN NARWHALS (MONODON MONOCEROS).

Karine Béland, Stéphane Lair, Mario Guay, Tristan Juette, Christian Bédard, Sandra R Black, Marianne Marcoux, Cortney A Watt, Nigel E Hussey, Marion Desmarchelier.

Narwhals (Monodon monoceros) are increasingly exposed to anthropogenic disturbances that may increase their stress levels with unknown consequences for the overall population dynamics. The validation and measurement of chronic stress biomarkers could contribute toward improved understanding and conservation efforts for this species. Dehydroepiandrosterone (DHEA) and its sulfated metabolite DHEA-S are collectively referred to as DHEA(S). Serum DHEA(S) concentrations combined in ratios with cortisol [cortisol/DHEA(S)] have been shown to be promising indicators of chronic stress in humans, domestic animals, and wildlife. During field tagging in 2017 and 2018 in Baffin Bay, Nunavut, Canada, 14 wild narwhals were sampled at the beginning and end of the capture-tagging procedures. Serum DHEA(S) were measured with commercially available competitive enzyme-linked immunosorbent assays (ELISA) developed for humans. A partial validation of the ELISA assays was performed by the determination of the intra-assay coefficient of variation, confirmation of the DHEA(S) dilutional linearity, and the calculation of the percentage of recovery. Mean values (nanograms per milliliter ± standard error of the mean) of narwhal serum cortisol, DHEA(S), and cortisol/DHEA(S) ratios, at the beginning and at the end of handling, respectively, are reported (cortisol = 30.74 ± 4.87 and 41.83 ± 4.83; DHEA = 1.01 ± 0.52 and 0.99 ± 0.50; DHEA-S = 8.72 ± 1.68 and 7.70 ± 1.02; cortisol/DHEA = 75.43 ± 24.35 and 84.41 ± 11.76, and cortisol/DHEA-S = 4.16 ± 1.07 and 6.14 ± 1.00). Serum cortisol and cortisol/DHEA-S were statistically higher at the end of the capture (P= 0.024 and P= 0.035, respectively). Moreover, serum cortisol at the end of handling was positively correlated to total body length (P = 0.042) and tended to be higher in males (P = 0.086). These assays proved easy to perform, rapid, and suitable for measuring serum DHEA(S) of narwhals and that calculated cortisol/DHEA(S) are potential biomarkers for chronic stress in narwhals and possibly other cetaceans.

DOI: https://doi.org/10.1638/2022-0049
Front Cell Dev Biol. 2023 ;11 957805

Tissue-specific expression of carbohydrate sulfotransferases drives keratan sulfate biosynthesis in the notochord and otic vesicles of Xenopus embryos.

Yuuri Yasuoka.

  Keratan sulfate (KS) is a glycosaminoglycan that is enriched in vertebrate cornea, cartilage, and brain. During embryonic development, highly sulfated KS (HSKS) is first detected in the developing notochord and then in otic vesicles; therefore, HSKS has been used as a molecular marker of the notochord. However, its biosynthetic pathways and functional roles in organogenesis are little known. Here, I surveyed developmental expression patterns of genes related to HSKS biosynthesis in Xenopus embryos. Of these genes, the KS chain-synthesizing glycosyltransferase genes, beta-1,3-N-acetylglucosaminyltransferase (b3gnt7) and beta-1,4-galactosyltransferase (b4galt4), are strongly expressed in the notochord and otic vesicles, but also in other tissues. In addition, their notochord expression is gradually restricted to the posterior end at the tailbud stage. In contrast, carbohydrate sulfotransferase (Chst) genes, chst2, chst3, and chst5.1, are expressed in both notochord and otic vesicles, whereas chst1, chst4/5-like, and chst7 are confined to otic vesicles. Because the substrate for Chst1 and Chst3 is galactose, while that for others is N-acetylglucosamine, combinatorial, tissue-specific expression patterns of Chst genes should be responsible for tissue-specific HSKS enrichment in embryos. As expected, loss of function of chst1 led to loss of HSKS in otic vesicles and reduction of their size. Loss of chst3 and chst5.1 resulted in HSKS loss in the notochord. These results reveal that Chst genes are critical for HSKS biosynthesis during organogenesis. Being hygroscopic, HSKS forms "water bags" in embryos to physically maintain organ structures. In terms of evolution, in ascidian embryos, b4galt and chst-like genes are also expressed in the notochord and regulate notochord morphogenesis. Furthermore, I found that a chst-like gene is also strongly expressed in the notochord of amphioxus embryos. These conserved expression patterns of Chst genes in the notochord of chordate embryos suggest that Chst is an ancestral component of the chordate notochord.

Keywords:  chordate bodyplan; evolution; gene duplication; morphogenesis; proteoglycan; subfunctionalization

DOI:  https://doi.org/10.3389/fcell.2023.957805
Biology (Basel). 2023 Mar 04. pii: 407. [Epub ahead of print]12(3):

Validation of Recombinant Heparan Sulphate Reagents for CNS Repair.

Susan L Lindsay, Rebecca Sherrard Smith, Edwin A Yates, Colin Cartwright, Bryan E Thacker, Jeremy E Turnbull, Charles A Glass, Susan C Barnett.

  Therapies that target the multicellular pathology of central nervous system (CNS) disease/injury are urgently required. Modified non-anticoagulant heparins mimic the heparan sulphate (HS) glycan family and have been proposed as therapeutics for CNS repair since they are effective regulators of numerous cellular processes. Our in vitro studies have demonstrated that low-sulphated modified heparan sulphate mimetics (LS-mHeps) drive CNS repair. However, LS-mHeps are derived from pharmaceutical heparin purified from pig intestines, in a supply chain at risk of shortages and contamination. Alternatively, cellular synthesis of heparin and HS can be achieved using mammalian cell multiplex genome engineering, providing an alternative source of recombinant HS mimetics (rHS). TEGA Therapeutics (San Diego) have manufactured rHS reagents with varying degrees of sulphation and we have validated their ability to promote repair in vitro using models that mimic CNS injury, making comparisons to LS-mHep7, a previous lead compound. We have shown that like LS-mHep7, low-sulphated rHS compounds promote remyelination and reduce features of astrocytosis, and in contrast, highly sulphated rHS drive neurite outgrowth. Cellular production of heparin mimetics may, therefore, offer potential clinical benefits for CNS repair.

Keywords:  CNS repair; heparan sulphate; neurite outgrowth; recombinant heparin mimetics; remyelination

DOI:  https://doi.org/10.3390/biology12030407
Viruses. 2023 Mar 01. pii: 663. [Epub ahead of print]15(3):

Heparan Sulfate and Enoxaparin Interact at the Interface of the Spike Protein of HCoV-229E but Not with HCoV-OC43.

Virginia Fuochi, Giuseppe Floresta, Rosalia Emma, Vincenzo Patamia, Massimo Caruso, Chiara Zagni, Federica Ronchi, Celestino Ronchi, Filippo Drago, Antonio Rescifina, Pio Maria Furneri.

  It is known that the spike protein of human coronaviruses can bind to a secondary receptor, or coreceptor, to facilitate the virus entry. While HCoV-229E uses human aminopeptidase N (hAPN) as a receptor, HCoV-OC43 binds to 9-O-acetyl-sialic acid (9-O-Ac-Sia), which is linked in a terminal way to the oligosaccharides that decorate glycoproteins and gangliosides on the surface of the host cell. Thus, evaluating the possible inhibitory activity of heparan sulfate, a linear polysaccharide found in animal tissues, and enoxaparin sodium on these viral strains can be considered attractive. Therefore, our study also aims to evaluate these molecules' antiviral activity as possible adsorption inhibitors against non-SARS-CoV. Once the molecules' activity was verified in in vitro experiments, the binding was studied by molecular docking and molecular dynamic simulations confirming the interactions at the interface of the spike proteins.

Keywords:  9-O-Ac-Sia; APN; HCoV-229E; HCoV-OC43; coronavirus; enoxaparin; heparan sulfate; molecular docking

DOI:  https://doi.org/10.3390/v15030663
Scand J Clin Lab Invest. 2023 Mar 29. 1-7

Sulfate and acid-base balance.

Troels Ring, Sebastian Frische, Stephen Edward Rees, Jette Nybo, Søren Risom Kristensen.

  It has been acknowledged for years that compounds containing sulfur (S) are an important source of endogenous acid production. In the metabolism, S is oxidized to sulfate, and therefore the mEq sulfate excreted in the urine is counted as acid retained in the body. In this study we show that pH in fluids with constant [Na] and [HEPES] declines as sulfate ions are added, and we show that titratable acidity increases exactly with the equivalents of sulfate. Therefore, sulfate excretion in urine is also acid excretion per se. This is in accordance with the down-regulation of proximal sulfate reabsorption under acidosis and the observation that children with distal renal tubular acidosis may be sulfate depleted. These results are well explained using charge-balance modeling, which is based only on the three fundamental principles of electroneutrality, conservation of mass, and rules of dissociation as devised from physical chemistry. In contrast, the findings are in contrast to expectations from conventional narratives. These are unable to understand the decreasing pH as sulfate is added since no conventional acid is present. The results may undermine the traditional notion of endogenous acid production since in the case of sulfur balance, S oxidation and its excretion as sulfate exactly balance each other. Possible clinical correlates with these findings are discussed.

Keywords:  Acid-base equilibrium; HEPES; biological; buffers; chemistry; computer simulations; electrochemistry; ions; models; physical; sulfates; water-electrolyte balance

DOI:  https://doi.org/10.1080/00365513.2023.2188607
Int J Mol Sci. 2023 Mar 10. pii: 5301. [Epub ahead of print]24(6):

DHEA and Its Metabolites Reduce the Cytokines Involved in the Inflammatory Response and Fibrosis in Primary Biliary Cholangitis.

Małgorzata Blatkiewicz, Katarzyna Sielatycka, Katarzyna Piotrowska, Ewa Kilańczyk.

  Dehydroepiandrosterone (DHEA) is an abundant steroid and precursor of sex hormones. During aging, the reduction in DHEA synthesis causes a significant depletion of estrogens and androgens in different organs, such as the ovaries, brain, and liver. Primary Biliary Cholangitis (PBC) is a cholestatic liver disease that begins with immune-mediated bile duct damage, and is followed by liver fibrosis, and finally, cirrhosis. PBC primarily affects postmenopausal women, with an average age of diagnosis of 65 years, but younger women are also affected. Here, we analyzed the levels of DHEA, estradiol (E2), and estriol (E3) in the PBC sera of females at an age of diagnosis under 40 (n = 37) and above 65 (n = 29). Our results indicate that in PBC patients at an age of diagnosis under 40, E2 was significantly lower compared to that in healthy women. In contrast, the levels of DHEA and E3 were in a normal range. Furthermore, ELISA assays revealed that in PBC patients at an age of diagnosis above 65, the levels of DHEA, E2, and E3 significantly declined in comparison to those in younger patients. In addition, flow cytometry analysis showed that the level of IL-8 significantly decreased while the level of TNF-α increased in older PBC patients compared to younger ones. Moreover, we showed for the first time that the sulfonated form of DHEA, DHEA-S, reduces the levels of both pro-inflammatory interleukins, IL-8 and TNF-α, in PBC-like cholangiocytes (H69-miR506), while it diminishes the level of the pro-fibrotic interleukin, IL-13, in hepatocytes (Hep-G2). Finally, we demonstrated that the expression of the pro-fibrotic agent TGF-β significantly increased in both the early (F0-F3) and cirrhotic (F4) stages of PBC, and this elevation was accompanied by higher α-SMA expression.

Keywords:  DHEA; PBC; estrogens; interleukins; steroidogenesis

DOI:  https://doi.org/10.3390/ijms24065301
J Med Chem. 2023 Mar 31.

Designing Smaller, Synthetic, Functional Mimetics of Sulfated Glycosaminoglycans as Allosteric Modulators of Coagulation Factors.

Elsamani I Abdelfadiel, Rama Gunta, Bharath Kumar Villuri, Daniel K Afosah, Nehru Viji Sankaranarayanan, Umesh R Desai.

Natural glycosaminoglycans (GAGs) are arguably the most diverse collection of natural products. Unfortunately, this bounty of structures remains untapped. Decades of research has realized only one GAG-like synthetic, small-molecule drug, fondaparinux. This represents an abysmal output because GAGs present a frontier that few medicinal chemists, and even fewer pharmaceutical companies, dare to undertake. GAGs are heterogeneous, polymeric, polydisperse, highly water soluble, synthetically challenging, too rapidly cleared, and difficult to analyze. Additionally, GAG binding to proteins is not very selective and GAG-binding sites are shallow. This Perspective attempts to transform this negative view into a much more promising one by highlighting recent advances in GAG mimetics. The Perspective focuses on the principles used in the design/discovery of drug-like, synthetic, sulfated small molecules as allosteric modulators of coagulation factors, such as antithrombin, thrombin, and factor XIa. These principles will also aid the design/discovery of sulfated agents against cancer, inflammation, and microbial infection.

DOI: https://doi.org/10.1021/acs.jmedchem.3c00132
Cells. 2023 Mar 16. pii: 910. [Epub ahead of print]12(6):

The Heparan Sulfate Proteoglycan Syndecan-1 Triggers Breast Cancer Cell-Induced Coagulability by Induced Expression of Tissue Factor.

Nourhan Hassan, Nico Bückreiß, Janes Efing, Marie Schulz-Fincke, Philipp König, Burkhard Greve, Gerd Bendas, Martin Götte.

  Syndecan-1 (Sdc-1) upregulation is associated with poor prognosis in breast cancer. Sdc-1 knockdown results in reduced angiogenesis and the dysregulation of tissue factor (TF) pathway constituents. Here, we evaluate the regulatory mechanisms and functional consequences of the Sdc-1/TF-axis using Sdc-1 knockdown and overexpression approaches in MCF-7 and MDA-MB-231 breast cancer cells. Gene expression was analyzed by means of qPCR. Thrombin generation and cell migration were detected. Cell-cycle progression and apoptosis were investigated using flow cytometry. In MDA-MB-231 cells, IL6, IL8, VEGF, and IGFR-dependent signaling affected TF pathway expression depending on Sdc-1. Notably, Sdc-1 depletion and TF pathway inhibitor (TFPI) synergistically affected PTEN, MAPK, and STAT3 signaling. At the functional level, the antiproliferative and pro-apoptotic effects of TFPI depended on Sdc-1, whereas Sdc-1's modulation of cell motility was not affected by TFPI. Sdc-1 overexpression in MCF-7 and MDA-MB-231 cells led to increased TF expression, inducing a procoagulative phenotype, as indicated by the activation of human platelets and increased thrombin formation. A novel understanding of the functional interplay between Sdc-1 and the TF pathway may be compatible with the classical co-receptor role of Sdc-1 in cytokine signaling. This opens up the possibility of a new functional understanding, with Sdc-1 fostering coagulation and platelet communication as the key to the hematogenous metastatic spread of breast cancer cells.

Keywords:  apoptosis; breast cancer; cell cycle; cell motility; heparan sulfate; platelets; signal transduction; syndecan-1; thrombin; tissue factor

DOI:  https://doi.org/10.3390/cells12060910
J Mol Cell Cardiol. 2023 Mar 24. pii: S0022-2828(23)00056-1. [Epub ahead of print]

Indoxyl-sulfate activation of the AhR- NF-κB pathway promotes interleukin-6 secretion and the subsequent osteogenic differentiation of human valvular interstitial cells from the aortic valve.

Alexandre Candellier, Nervana Issa, Maria Grissi, Théo Brouette, Carine Avondo, Cathy Gomila, Gérémy Blot, Brigitte Gubler, Gilles Touati, Youssef Bennis, Thierry Caus, Michel Brazier, Gabriel Choukroun, Christophe Tribouilloy, Saïd Kamel, Cédric Boudot, Lucie Hénaut.

  BACKGROUND: Calcific aortic stenosis (CAS) is more prevalent, occurs earlier, progresses faster and has worse outcomes in patients with chronic kidney disease (CKD). The uremic toxin indoxyl sulfate (IS) is powerful predictor of cardiovascular mortality in these patients and a strong promoter of ectopic calcification whose role in CAS remains poorly studied. The objective of this study was to evaluate whether IS influences the mineralization of primary human valvular interstitial cells (hVICs) from the aortic valve.METHODS: Primary hVICs were exposed to increasing concentrations of IS in osteogenic medium (OM). The hVICs' osteogenic transition was monitored by qRT-PCRs for BMP2 and RUNX2 mRNA. Cell mineralization was assayed using the o-cresolphthalein complexone method. Inflammation was assessed by monitoring NF-κB activation using Western blots as well as IL-1β, IL-6 and TNF-α secretion by ELISAs. Small interfering RNA (siRNA) approaches enabled us to determine which signaling pathways were involved.
RESULTS: Indoxyl-sulfate increased OM-induced hVICs osteogenic transition and calcification in a concentration-dependent manner. This effect was blocked by silencing the receptor for IS (the aryl hydrocarbon receptor, AhR). Exposure to IS promoted p65 phosphorylation, the blockade of which inhibited IS-induced mineralization. Exposure to IS promoted IL-6 secretion by hVICs, a phenomenon blocked by silencing AhR or p65. Incubation with an anti-IL-6 antibody neutralized IS's pro-calcific effects.
CONCLUSION: IS promotes hVIC mineralization through AhR-dependent activation of the NF-κB pathway and the subsequent release of IL-6. Further research should seek to determine whether targeting inflammatory pathways can reduce the onset and progression of CKD-related CAS.

Keywords:  aortic stenosis; indoxyl sulfate; inflammation; valvular interstitial cell

DOI:  https://doi.org/10.1016/j.yjmcc.2023.03.011