Peripheral blood syndecan-1 levels after mechanical thrombectomy can predict the clinical prognosis of patients with acute ischemic stroke.

BACKGROUND: Previously, we revealed noticeable dynamic fluctuations in syndecan-1 levels in the peripheral blood of post-stroke patients. We further investigated the clinical prognostic value of syndecan-1 as a biomarker of glycoprotein damage in patients with acute ischaemic stroke (AIS). METHODS: We examined 105 patients with acute large vessel occlusion in the anterior circulation, all of whom underwent mechanical thrombectomy (MT). Peripheral blood syndecan-1 levels were measured 1 day after MT, and patients were categorised into favourable and unfavourable prognostic groups based on the 90-day modified Rankin Scale (mRS) score. Additionally, we compared the clinical outcomes between groups with high and low syndecan-1 concentrations. RESULTS: The findings revealed a significantly lower syndecan-1 level in the group with an unfavourable prognosis compared to those with a favourable prognosis (p < 0.01). In the multivariable logistic regression analysis, lower syndecan-1 levels were identified as a predictor of unfavourable prognosis (odds ratio (OR) = 0.965, p = 0.001). Patients displaying low syndecan-1 expression in the peripheral blood (< 29.51 ng/mL) experienced a > twofold increase in the rates of unfavourable prognosis and mortality. CONCLUSIONS: Our study demonstrates that syndecan-1, as an emerging, easily detectable stroke biomarker, can predict the clinical outcomes of patients with AIS. After MT, low levels of syndecan-1 in the peripheral blood on the first day emerged as an independent risk factor for an unfavourable prognosis, suggesting that lower syndecan-1 levels might signify worse clinical presentation and outcomes in stroke patients undergoing this procedure.

Intramuscular Exposure to a Lethal Dose of Ricin Toxin Leads to Endothelial Glycocalyx Shedding and Microvascular Flow Abnormality in Mice and Swine.

Ricin toxin isolated from the castor bean (Ricinus communis) is one of the most potent and lethal molecules known. While the pathophysiology and clinical consequences of ricin poisoning by the parenteral route, i.e., intramuscular penetration, have been described recently in various animal models, the preceding mechanism underlying the clinical manifestations of systemic ricin poisoning has not been completely defined. Here, we show that following intramuscular administration, ricin bound preferentially to the vasculature in both mice and swine, leading to coagulopathy and widespread hemorrhages. Increased levels of circulating VEGF and decreased expression of vascular VE-cadherin caused blood vessel impairment, thereby promoting hyperpermeability in various organs. Elevated levels of soluble heparan sulfate, hyaluronic acid and syndecan-1 were measured in blood samples following ricin intoxication, indicating that the vascular glycocalyx of both mice and swine underwent extensive damage. Finally, by using side-stream dark field intravital microscopy imaging, we determined that ricin poisoning leads to microvasculature malfunctioning, as manifested by aberrant blood flow and a significant decrease in the number of diffused microvessels. These findings, which suggest that glycocalyx shedding and microcirculation dysfunction play a major role in the pathology of systemic ricin poisoning, may serve for the formulation of specifically tailored therapies for treating parenteral ricin intoxication.

In vivo evaluation of CD38 and CD138 as targets for nanoparticle-based drug delivery in multiple myeloma.

BACKGROUND: Drug-loaded nanoparticles have established their benefits in the fight against multiple myeloma; however, ligand-targeted nanomedicine has yet to successfully translate to the clinic due to insufficient efficacies reported in preclinical studies. METHODS: In this study, liposomal nanoparticles targeting multiple myeloma via CD38 or CD138 receptors are prepared from pre-synthesized, purified constituents to ensure increased consistency over standard synthetic methods. These nanoparticles are then tested both in vitro for uptake to cancer cells and in vivo for accumulation at the tumor site and uptake to tumor cells. Finally, drug-loaded nanoparticles are tested for long-term efficacy in a month-long in vivo study by tracking tumor size and mouse health. RESULTS: The targeted nanoparticles are first optimized in vitro and show increased uptake and cytotoxicity over nontargeted nanoparticles, with CD138-targeting showing superior enhancement over CD38-targeted nanoparticles. However, biodistribution and tumor suppression studies established CD38-targeted nanoparticles to have significantly increased in vivo tumor accumulation, tumor cell uptake, and tumor suppression over both nontargeted and CD138-targeted nanoparticles due to the latter's poor selectivity. CONCLUSION: These results both highlight a promising cancer treatment option in CD38-targeted nanoparticles and emphasize that targeting success in vitro does not necessarily translate to success in vivo.

Synthesis of O-Sulfated Human Syndecan-1-like Glyco-polypeptides by Incorporating Peptide Ligation and O-Sulfated Glycopeptide Cassette Strategies.

A successful synthesis of O-sulfated syndecan-1-like (Q23-E120) glyco-polypeptide was accomplished. The synthesis features the integration of an O-sulfated carbohydrate-bearing glycopeptide cassette with efficient protein ligation strategies, overcoming the acid lability of carbohydrate sulfates as a major hurdle in solid-phase peptide synthesis. Crucial to the synthesis is the microwave-assisted Ag(I) ligation, which afforded the ligation product in improved overall yield. This O-sulfated syndecan-1 (Q23-E120) is the longest O-sulfated glyco-polypeptide prepared to date.

Heparin and heparan sulfate proteoglycans promote HIV-1 p17 matrix protein oligomerization: computational, biochemical and biological implications.

p17 matrix protein released by HIV+ cells interacts with leukocytes heparan sulfate proteoglycans (HSPGs), CXCR1 and CXCR2 exerting different cytokine-like activities that contribute to AIDS pathogenesis. Since the bioactive form of several cytokines is represented by dimers/oligomers and oligomerization is promoted by binding to heparin or HSPGs, here we evaluated if heparin/HSPGs also promote p17 oligomerization. Heparin favours p17 dimer, trimer and tetramer assembly, in a time- and biphasic dose-dependent way. Heparin-induced p17 oligomerization is of electrostatic nature, being it prevented by NaCl, by removing negative sulfated groups of heparin and by neutralizing positive lysine residues in the p17 N-terminus. A new computational protocol has been implemented to study heparin chains up to 24-mer accommodating a p17 dimer. Molecular dynamics show that, in the presence of heparin, two p17 molecules undergo conformational modifications creating a continuous "electropositive channel" in which heparin sulfated groups interact with p17 basic amino acids, promoting its dimerization. At the cell surface, HSPGs induce p17 oligomerization, as demonstrated by using B-lymphoblastoid Namalwa cells overexpressing the HSPG Syndecan-1. Also, HSPGs on the surface of BJAB and Raji human B-lymphoblastoid cells are required to p17 to induce ERK1/2 activation, suggesting that HS-induced oligomerization plays a role in p17-induced lymphoid dysregulation during AIDS.

Dynamics of soluble syndecan-1 in maternal serum during and after pregnancies complicated by preeclampsia: a nested case control study.

Background We investigated the dynamics and the predictive value of soluble syndecan-1 (Sdc-1), a biomarker of endothelial dysfunction, in uneventful pregnancies and pregnancies complicated by preeclampsia (PE). Methods Serum levels of Sdc-1 were measured at sequential time points during and after uneventful pregnancies (control, n = 95) and pregnancies developing PE (PE_long, n = 12). Levels were further measured in women with symptomatic PE (PE_state, n = 46) at a single time point. Results Sdc-1 levels increased consistently throughout pregnancy. In the PE_long group Sdc-1 levels were lower at all visits throughout pregnancy, and reached significance in weeks 18-22 (p = 0.019), 23-27 (p = 0.009), 28-32 (p = 0.006) and 33-36 (p = 0.008). After delivery, Sdc-1 levels dropped sharply in all pregnancies but were significantly elevated in the PE_long group. The predictive power of Sdc-1 was evaluated analyzing receiver operating characteristic (ROC) curves. A significant power was reached at weeks 14-17 (area under the curve [AUC] 0.65, p = 0.025), 23-27 (AUC 0.73, p = 0.004) and 33-36 (AUC 0.75, p = 0.013). Conclusions In summary, Sdc-1 levels were lower in women developing PE compared to uneventful pregnancies and Sdc-1 might be useful to predict PE. After delivery, Sdc-1 levels remained higher in women with PE. Additional studies investigating the link between glycocalyx degradation, Sdc-1 levels and placental and endothelial dysfunction in pregnancies affected by PE are warranted.

Syndecan-1 Mediates Sorting of Soluble Lipoprotein Lipase with Sphingomyelin-Rich Membrane in the Golgi Apparatus.

In the secretory pathway, budding of vesicular transport carriers from the trans-Golgi network (TGN) must coordinate specification of lipid composition with selection of secreted proteins. We elucidate a mechanism of soluble protein cargo sorting into secretory vesicles with a sphingomyelin-rich membrane; the integral membrane proteoglycan Syndecan-1 (SDC1) acts as a sorting receptor, capturing the soluble enzyme lipoprotein lipase (LPL) during export from the TGN. Sorting of LPL requires bivalent interactions between LPL and SDC1-linked heparan sulfate chains and between LPL and the Golgi membrane. Physical features of the SDC1 transmembrane domain, rather than a specific sequence, confer targeting of SDC1 and bound LPL into the sphingomyelin secretion pathway. This study establishes that physicochemical properties of a protein transmembrane domain that drive lateral heterogeneity of the plasma membrane also operate at the TGN to confer sorting of an integral membrane protein and its ligand within the biosynthetic secretory pathway.

What is the Best Radionuclide for Immuno-PET of Multiple Myeloma? A Comparison Study Between 89Zr- and 64Cu-Labeled Anti-CD138 in a Preclinical Syngeneic Model.

Although positron emission tomography (PET) imaging with 18-Fluorodeoxyglucose (18F-FDG) is a promising technique in multiple myeloma (MM), the development of other radiopharmaceuticals seems relevant. CD138 is currently used as a standard marker for the identification of myeloma cells and could be used in phenotype tumor imaging. In this study, we used an anti-CD138 murine antibody (9E7.4) radiolabeled with copper-64 (64Cu) or zirconium-89 (89Zr) and compared them in a syngeneic mouse model to select the optimal tracers for MM PET imaging. Then, 9E7.4 was conjugated to TE2A-benzyl isothiocyanate (TE2A) and desferrioxamine (DFO) chelators for 64Cu and 89Zr labeling, respectively. 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 antibodies were evaluated by PET imaging and biodistribution studies in C57BL/KaLwRij mice bearing either 5T33-MM subcutaneous tumors or bone lesions and were compared to 18F-FDG-PET imaging. In biodistribution and PET studies, 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 displayed comparable good tumor uptake of subcutaneous tumors. On the bone lesions, PET imaging with 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 showed higher uptake than with 18F-FDG-PET. Comparison of both 9E7.4 conjugates revealed higher nonspecific bone uptakes of 89Zr-DFO-9E7.4 than 64Cu-TE2A-9E7.4. Because of free 89Zr's tropism for bone when using 89Zr-anti-CD138, 64Cu-anti-CD138 antibody had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a specific new imaging radiopharmaceutical agent in MM.

Adaptive landscape flattening in amino acid sequence space for the computational design of protein:peptide binding.

For the high throughput design of protein:peptide binding, one must explore a vast space of amino acid sequences in search of low binding free energies. This complex problem is usually addressed with either simple heuristic scoring or expensive sequence enumeration schemes. Far more efficient than enumeration is a recent Monte Carlo approach that adaptively flattens the energy landscape in sequence space of the unbound peptide and provides formally exact binding free energy differences. The method allows the binding free energy to be used directly as the design criterion. We propose several improvements that allow still more efficient sampling and can address larger design problems. They include the use of Replica Exchange Monte Carlo and landscape flattening for both the unbound and bound peptides. We used the method to design peptides that bind to the PDZ domain of the Tiam1 signaling protein and could serve as inhibitors of its activity. Four peptide positions were allowed to mutate freely. Almost 75 000 peptide variants were processed in two simulations of 109 steps each that used 1 CPU hour on a desktop machine. 96% of the theoretical sequence space was sampled. The relative binding free energies agreed qualitatively with values from experiment. The sampled sequences agreed qualitatively with an experimental library of Tiam1-binding peptides. The main assumption limiting accuracy is the fixed backbone approximation, which could be alleviated in future work by using increased computational resources and multi-backbone designs.

Accurate PDZ/Peptide Binding Specificity with Additive and Polarizable Free Energy Simulations.

PDZ domains contain 80-100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.

Syndecan-1 in mechanosensing of nanotopological cues in engineered materials.

The cells of the vascular system are highly sensitive to biophysical cues from their local cellular microenvironment. To engineer improved materials for vascular devices and delivery of cell therapies, a key challenge is to understand the mechanisms that cells use to sense biophysical cues from their environment. Syndecans are heparan sulfate proteoglycans (HSPGs) that consist of a protein core modified with heparan sulfate glycosaminoglycan chains. Due to their presence on the cell surface and their interaction with cytoskeletal and focal adhesion associated molecules, cell surface proteoglycans are well poised to serve as mechanosensors of the cellular microenvironment. Nanotopological cues have become recognized as major regulators of cell growth, migration and phenotype. We hypothesized that syndecan-1 could serve as a mechanosensor for nanotopological cues and can mediate the responsiveness of vascular smooth muscle cells to nanoengineered materials. We created engineered substrates made of polyurethane acrylate with nanogrooves using ultraviolet-assisted capillary force lithography. We cultured vascular smooth muscle cells with knockout of syndecan-1 on engineered substrates with varying compliance and nanotopology. We found that knockout of syndecan-1 reduced alignment of vascular smooth muscle cells to the nanogrooves under inflammatory treatments. In addition, we found that loss of syndecan-1 increased nuclear localization of Yap/Taz and phospho-Smad2/3 in response to nanogrooves. Syndecan-1 knockout vascular smooth muscle cells also had elevated levels of Rho-associated protein kinase-1 (Rock1), leading to increased cell stiffness and an enhanced contractile state in the cells. Together, our findings support that syndecan-1 knockout leads to alterations in mechanosensing of nanotopographical cues through alterations of in rho-associated signaling pathways, cell mechanics and mediators of the Hippo and TGF-ß signaling pathways.

Harnessing a catalytic lysine residue for the one-step preparation of homogeneous antibody-drug conjugates.

Current strategies to produce homogeneous antibody-drug conjugates (ADCs) rely on mutations or inefficient conjugation chemistries. Here we present a strategy to produce site-specific ADCs using a highly reactive natural buried lysine embedded in a dual variable domain (DVD) format. This approach is mutation free and drug conjugation proceeds rapidly at neutral pH in a single step without removing any charges. The conjugation chemistry is highly robust, enabling the use of crude DVD for ADC preparation. In addition, this strategy affords the ability to precisely monitor the efficiency of drug conjugation with a catalytic assay. ADCs targeting HER2 were prepared and demonstrated to be highly potent and specific in vitro and in vivo. Furthermore, the modular DVD platform was used to prepare potent and specific ADCs targeting CD138 and CD79B, two clinically established targets overexpressed in multiple myeloma and non-Hodgkin lymphoma, respectively.

Glycosaminoglycans and glycolipids as potential biomarkers in lung cancer.

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.

Extracellularly Extruded Syntaxin-4 Binds to Laminin and Syndecan-1 to Regulate Mammary Epithelial Morphogenesis.

Epithelial morphogenesis in the mammary gland proceeds as a consequence of complex cell behaviors including apoptotic cell death and epithelial-mesenchymal transition (EMT); the extracellular matrix (ECM) protein laminin is crucially involved. Syntaxins mediate intracellular vesicular fusion, yet certain plasmalemmal members have been shown to possess latent extracellular functions. In this study, the extracellular subpopulation of syntaxin-4, extruded in response to the induction of differentiation or apoptosis in mammary epithelial cells, was detected. Using a tetracycline-repressive transcriptional system and clonal mammary epithelial cells, SCp2, we found that the expression of cell surface syntaxin-4 elicits EMT-like cell behaviors. Intriguingly, these cells did not up-regulate key transcription factors associated with the canonical EMT such as snail, slug, or twist, and repressed translation of E-cadherin. Concurrently, the cells completely evaded the cellular aggregation/rounding triggered by a potent EMT blocker laminin-111. We found that the recombinant form of syntaxin-4 not only bound to laminin but also latched onto the glycosaminoglycan (GAG) side chains of syndecan-1, a laminin receptor that mediates epithelial morphogenesis. Thus, temporal extracellular extrusion of syntaxin-4 emerged as a novel regulatory element for laminin-induced mammary epithelial cell behaviors. J. Cell. Biochem. 118: 686-698, 2017. © 2016 Wiley Periodicals, Inc.

Tumor specific liposomes improve detection of pancreatic adenocarcinoma in vivo using optoacoustic tomography.

BACKGROUND: Pancreatic cancer often goes undiagnosed until late stage disease due in part to suboptimal early detection. Our goal was to develop a Syndecan-1 tagged liposome containing fluorescent dye as an improved contrast agent for detection of pancreatic adenocarcinoma in vivo using multispectral optoacoustic tomography. RESULTS: The diagnostic capabilities and specificity to pancreatic adenocarcinoma of Syndecan-1 targeted liposomes were evaluated both in vitro and in vivo. Immunocytochemistry showed that liposomes preferentially bound to and released their contents into cells expressing high levels of insulin-like growth factor 1 receptor. We determined that the contents of the liposome were released into the cell as noted by the change in propidium iodide fluorescence from green to red based upon nucleic acid binding. In an orthotopic mouse model, the liposomes preferentially targeted the pancreatic tumor with little off-target binding in the liver and spleen. Peak accumulation of the liposomes in the tumor occurred at 8 h post-injection. Multispectral optoacoustic tomographic imaging was able to provide high-resolution 3D images of the tumor and liposome location. Ex vivo analysis showed that non-targeted liposomes accumulated in the liver, suggesting that specificity of the liposomes for pancreatic adenocarcinoma was due to the presence of the Syndecan-1 ligand. CONCLUSIONS: This study demonstrated that Syndecan-1 liposomes were able to release cargo into IGF1-R expressing tumor cells. The Syndecan-1 liposomes demonstrated tumor specificity in orthotopic pancreatic cancer as observed using multispectral optoacoustic tomography with reduced kidney and liver uptake. By targeting the liposome with Syndecan-1, this nanovehicle has potential as a targeted theranostic nanoparticle for both drug and contrast agent delivery to pancreatic tumors.

Syndecan-1 and Syndecan-4 Capture Epidermal Growth Factor Receptor Family Members and the α3ß1 Integrin Via Binding Sites in Their Ectodomains: NOVEL SYNSTATINS PREVENT KINASE CAPTURE AND INHIBIT α6ß4-INTEGRIN-DEPENDENT EPITHELIAL CELL MOTILITY.

The α6ß4 integrin is known to associate with receptor tyrosine kinases when engaged in epithelial wound healing and in carcinoma invasion and survival. Prior work has shown that HER2 associates with α6ß4 integrin and syndecan-1 (Sdc1), in which Sdc1 engages the cytoplasmic domain of the ß4 integrin subunit allowing HER2-dependent motility and carcinoma cell survival. In contrast, EGFR associates with Sdc4 and the α6ß4 integrin, and EGFR-dependent motility depends on cytoplasmic engagement of ß4 integrin with Sdc4. However, how HER2 and EGFR assimilate into a complex with the syndecans and integrin, and why kinase capture is syndecan-specific has remained unknown. In the present study, we demonstrate that HER2 is captured via a site, comprised of amino acids 210-240, in the extracellular domain of human Sdc1, and EGFR is captured via an extracellular site comprised of amino acids 87-131 in human Sdc4. Binding assays using purified recombinant proteins demonstrate that the interaction between the EGFR family members and the syndecans is direct. The α3ß1 integrin, which is responsible for the motility of the cells, is captured at these sites as well. Peptides based on the interaction motifs in Sdc1 and Sdc4, called synstatins (SSTN210-240 and SSTN87-131) competitively displace the receptor tyrosine kinase and α3ß1 integrin from the syndecan with an IC50 of 100-300 nm. The syndecans remain anchored to the α6ß4 integrin via its cytoplasmic domain, but the activation of cell motility is disrupted. These novel SSTN peptides are potential therapeutics for carcinomas that depend on these HER2- and EGFR-coupled mechanisms for their invasion and survival.

S-maltoheptaose targets syndecan-bound effectors to reduce smoking-related neutrophilic inflammation.

Cigarette smoke induces injury and neutrophilic inflammation in the airways of smokers. The stability and activity of inflammatory effectors, IL8 and neutrophil elastase (NE), can be prolonged by binding to airway heparan sulfate (HS)/syndecan-1, posing risk for developing chronic obstructive pulmonary disease(COPD). We hypothesize that antagonizing HS/syndecan-1 binding of the inflammatory effectors could reduce smoking-related neutrophil-mediated airway inflammation. Analysis of bronchoalveolar lavage fluid(BALF) of COPD patients found both total and unopposed NE levels to be significantly higher among smokers with COPD than non-COPD subjects. Similar NE burden was observed in smoke-exposed rats compared to sham air controls. We chose sulfated-maltoheptaose(SM), a heparin-mimetic, to antagonize HS/sydecan-1 binding of the inflammatory mediators in airway fluids and lung tissues of the smoke-exposed rat model. Airway treatment with SM resulted in displacement of CINC-1 and NE from complexation with bronchio-epithelial HS/syndecan-1, dissipating the chemokine gradient for neutrophil flux across to the bronchial lumen. Following SM displacement of NE from shed HS/syndecan-1 in bronchial fluids, NE became accessible to inhibition by α1-antitrypsin endogenous in test samples. The antagonistic actions of SM against syndecan-1 binding of NE and CINC-1 in smoke-exposed airways suggest new therapeutic opportunities for modulating airway inflammation in smokers with SM delivery.

Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation.

Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.

2-O-Sulfated Domains in Syndecan-1 Heparan Sulfate Inhibit Neutrophil Cathelicidin and Promote Staphylococcus aureus Corneal Infection.

Ablation of syndecan-1 in mice is a gain of function mutation that enables mice to significantly resist infection by several bacterial pathogens. Syndecan-1 shedding is induced by bacterial virulence factors, and inhibition of shedding attenuates bacterial virulence, whereas administration of purified syndecan-1 ectodomain enhances virulence, suggesting that bacteria subvert syndecan-1 ectodomains released by shedding for their pathogenesis. However, the pro-pathogenic functions of syndecan-1 ectodomain have yet to be clearly defined. Here, we examined how syndecan-1 ectodomain enhances Staphylococcus aureus virulence in injured mouse corneas. We found that syndecan-1 ectodomain promotes S. aureus corneal infection in an HS-dependent manner. Surprisingly, we found that this pro-pathogenic activity is dependent on 2-O-sulfated domains in HS, indicating that the effects of syndecan-1 ectodomain are structure-based. Our results also showed that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate motifs inhibit S. aureus killing by antimicrobial factors secreted by degranulated neutrophils, but does not affect intracellular phagocytic killing by neutrophils. Immunodepletion of antimicrobial factors with staphylocidal activities demonstrated that CRAMP, a cationic antimicrobial peptide, is primarily responsible for S. aureus killing among other factors secreted by degranulated neutrophils. Furthermore, we found that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate units potently and specifically inhibit S. aureus killing by synthetic CRAMP. These results provide compelling evidence that a specific subclass of sulfate groups, and not the overall charge of HS, permits syndecan-1 ectodomains to promote S. aureus corneal infection by inhibiting a key arm of neutrophil host defense.

A transmembrane C-terminal fragment of syndecan-1 is generated by the metalloproteinase ADAM17 and promotes lung epithelial tumor cell migration and lung metastasis formation.

Syndecan-1 is a heparan sulfate proteoglycan expressed by endothelial and epithelial cells and involved in wound healing and tumor growth. Surface-expressed syndecan-1 undergoes proteolytic shedding leading to the release of the soluble N-terminal ectodomain from a transmembrane C-terminal fragment (tCTF). We show that the disintegrin and metalloproteinase (ADAM) 17 generates a syndecan-1 tCTF, which can then undergo further intra-membrane proteolysis by γ-secretase. Scratch-induced wound closure of cultured lung epithelial A549 tumor cells associates with increased syndecan-1 cleavage as evidenced by the release of shed syndecan-1 ectodomain and enhanced generation of the tCTF. Both wound closure and the associated syndecan-1 shedding can be suppressed by inhibition of ADAM family proteases. Cell proliferation, migration and invasion into matrigel as well as several signaling pathways implicated in these responses are suppressed by silencing of syndecan-1. These defects of syndecan-1 deficient cells can be overcome by overexpression of syndecan-1 tCTF or a corresponding tCTF of syndecan-4 but not by overexpression of a tCTF lacking the transmembrane domain. Finally, lung metastasis formation of A549 cells in SCID mice was found to be dependent on syndecan-1, and the presence of syndecan-1 tCTF was sufficient for this activity. Thus, the syndecan-1 tCTF by itself is capable of mediating critical syndecan-1-dependent functions in cell proliferation, migration, invasion and metastasis formation and therefore can replace full length syndecan-1 in the situation of increased syndecan-1 shedding during cell migration and tumor formation.