Endogenous Glycosaminoglycans Research Articles

Endogenous Dysregulation of Thromboinflammatory Biomarkers in End-Stage Renal Disease, and Their Amplification by Heart Failure.

In patients with end-stage renal disease (ESRD), heart failure with reduced ejection fraction (HFrEF) is a common comorbidity. Thromboinflammatory processes in both conditions represent complex pathophysiology, demonstrated by dysregulation of thromboinflammatory biomarkers, and commonly resulting in the combined pathology of cardiorenal syndrome. We sought to investigate the effects of HFrEF on these biomarkers in patients with ESRD, and observe the relationship to mortality. Blood samples from 73 patients with ESRD (mean age 67 ± 13 years, 56% male) and 40 healthy controls were analyzed via enzyme-linked immunosorbent assay and other chromogenic methods for angiopoietin-2 (Ang2), endogenous glycosaminoglycans, fatty acid binding protein, interleukin-6, lipopolysaccharide, free fatty acids, NT-pro B-type natriuretic peptide, tumor necrosis factor α, vascular endothelial growth factor, and von Willebrand factor. Patients were stratified into those with or without HFrEF (EF < 50%). Patients had highly prevalent comorbidities including coronary artery disease 46%, diabetes 69%, hypertension 97%, and smoking 49%. Most biomarkers were upregulated in ESRD compared to controls. Patients with HFrEF and ESRD had greater interleukin-6 and NT-pro B-type natriuretic peptide and lesser lipopolysaccharide compared to ESRD only. Spearman correlations between most biomarkers were increased in HFrEF + ESRD over ESRD only. Ang-2 was associated with mortality in this cohort. The dysregulation of thromboinflammation in ESRD is somewhat amplified in comorbid HFrEF. Correlation among biomarkers in this cohort indicates the mechanisms of thromboinflammatory biomarker generation in ESRD and HFrEF share an integrative process. Ang2, interleukin-6, and lipopolysaccharide show promise as biomarkers for risk stratification among patients with both HFrEF and ESRD.

Fluid restrictive resuscitation with high molecular weight hyaluronan infusion in early peritonitis sepsis

Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cannot be discarded based on the results of the present study.

#2728 THROMBOINFLAMMATORY BIOMARKERS AND THEIR RELATIONSHIP WITH CIRCULATING GLYCOSAMINOGLYCANS IN END-STAGE RENAL DISEASE PATIENTS

Abstract Background and Aims End stage renal disease (ESRD) is a complex progressive medical condition that affects multiple organ systems. Given the high risk of morbidity and mortality of ESRD as well as its rising incidence, it is critical to understand the relevance of thromboinflammatory biomarkers in disease development and progression of kidney dysfunction. The purpose of this study is to profile the levels of thromboinflammatory biomarkers in ESRD patients including D-Dimer, C-reactive protein (CRP), von Willebrand factor (vWF), plasminogen activator inhibitor 1 (PAI-1), and thrombin activatable fibrinolysis inhibitor (TAFI). In addition, the levels of anti-PF4 IgG and endogenous circulating glycosaminoglycans (GAGs) were measured. Method Citrated plasma samples were collected from seventy-three ESRD patients. Control plasma samples (NHP) from healthy, non-smoking adults aged 19 to 53 were obtained commercially. Validated ELISA methods have been used to profile each of the biomarkers. The levels of endogenous GAGs were determined (Redprobes UG, Germany). To compare the levels of thromboinflammatory biomarkers, anti-PF4 IgG, and endogenous GAGs in different groups, appropriate statistical methods included Mann-Whitney U, t-tests, Kruskal-Wallis ANOVA and experiment correlation analysis methods were performed. Results All of the biomarkers and GAGs were significantly elevated, with the exception of TAFI, in ESRD patients. The ESRD patients exhibited varying levels of increase in the D-Dimer, CRP, vWF, PAI-1, anti-PF4 IgG, and GAGs as shown in Figure 1 (p &amp;lt; 0.05). D-Dimer showed the most pronounced increase (1075%) followed by PAI-1 (361.31%), anti-PF4 IgG (209.78%), CRP (101.77%) and endogenous GAGs (17.29%). The correlation analysis revealed varying degrees of association among these biomarkers (Figure 2). Conclusion These results suggest that thromboinflammatory biomarkers offer the potential utility of identifying inflammation in end-stage renal disease. Marked increase in thromboinflammatory mediators due to endothelial damage may result in the upregulation of glycosaminoglycans and anti-PF4 IgG antibodies in the ESRD patients.

HIGH-MOLECULAR-WEIGHT HYALURONAN-A POTENTIAL ADJUVANT TO FLUID RESUSCITATION IN ABDOMINAL SEPSIS?

While fluid resuscitation is fundamental in the treatment of sepsis-induced tissue hypoperfusion, a sustained positive fluid balance is associated with excess mortality. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water, has not been tested previously as adjuvant to fluid resuscitation in sepsis. In a prospective, parallel-grouped, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with adjuvant hyaluronan (add-on to standard therapy, n = 8) or 0.9% saline (n = 8). After the onset of hemodynamic instability, the animals received an initial bolus of 0.1% hyaluronan (1 mg/kg/10 min) or placebo (0.9% saline) followed by a continuous infusion of 0.1% hyaluronan (1 mg/kg/h) or saline during the experiment. We hypothesized that the administration of hyaluronan would reduce the volume of fluid administered (aiming at stroke volume variation <13%) and/or attenuate the inflammatory reaction. Total volumes of intravenous fluids infused were 17.5 ± 11 versus 19.0 ± 7 mL/kg/h in intervention and control groups, respectively ( P = 0.442). Plasma IL-6 increased to 2,450 (1,420-6,890) pg/mL and 3,690 (1,410-11,960) pg/mL (18 hours of resuscitation) in the intervention and control groups (nonsignificant). The intervention counteracted the increase in proportion of fragmented hyaluronan associated with peritonitis sepsis (mean peak elution fraction [18 hours of resuscitation] intervention group: 16.8 ± 0.9 versus control group: 17.9 ± 0.6 [ P = 0.031]). In conclusion, hyaluronan did not reduce the volume needed for fluid resuscitation or decrease the inflammatory reaction, even though it counterbalanced the peritonitis-induced shift toward increased proportion of fragmented hyaluronan.

Glycemic Control and Plasma Levels of Pro-Inflammatory and Pro-Thrombotic Biomarkers in Diabetic Patients Presenting with Acute Pulmonary Embolism.

Type I and type II diabetes are closely associated with a pro-inflammatory state and to a pro-thrombotic state. The role of glycemic control in pulmonary embolism (PE) is poorly understood and requires additional investigation. The aim of this study is to investigate the relationship between glycemic control and thrombo-inflammatory biomarkers in a PE patient cohort compared to normal samples. Demographic and clinical information for 86 diabetic patients and 106 non-diabetic patients presenting with acute PE was collected via retrospective chart review. Plasma levels of pro-inflammatory (C-reactive protein [CRP], tumor necrosis factor-alpha [TNF-α], interleukin-6 [IL-6]) and pro-thrombotic (d-dimer, plasminogen activator inhibitor-1 [PAI-1], tissue plasminogen activator [tPA], thrombin activatable fibrinolysis inhibitor [TAFI], von-Willebrand factor [vWF], endogenous glycosaminoglycans [GAGs]) biomarkers were drawn within 24 hours of diagnosis of acute PE. Data was also obtained for a population of healthy adult controls. All the pro-inflammatory and pro-thrombotic biomarkers were elevated in diabetic PE patients in comparison to healthy controls. None of the biomarkers were elevated in diabetic PE patients when compared to non-diabetic PE patients. There was no difference in the levels of the pro-inflammatory biomarkers according to glycemic control. The plasma level of TAFI was elevated in diabetic patients with poor glycemic control. Diabetic patients were more likely to have a more severe PE. These studies demonstrate that thrombo-inflammatory biomarkers are elevated in diabetic PE patients with associated comorbidities in comparison to normal individuals. However, there is no difference between the PE cohort alone in comparison to PE with diabetes. The role of TAFI within the continuum of diabetic vascular disease warrants additional investigation.

The Relevance of Thrombo-Inflammatory Biomarkers and Their Relationship with Circulating Glycosaminoglycans in End-Stage Renal Disease Patients

En-stage renal disease (ESRD) is a growing public health problem. The atherosclerotic cardiovascular complications are the leading causes of mortality and morbidity in the ESRD. In this study, we sought to quantify the levels of thrombo-inflammatory biomarkers in an ESRD patients in comparison to healthy controls to determine their relevance in thrombo-inflammation and adverse outcomes. The levels of D-Dimer, C-reactive protein (CRP), plasminogen activator inhibitor 1 (PAI-1) antigen, functional PAI-1, thrombin activatable fibrinolysis inhibitor, tissue plasminogen activator, von Willebrand factor, and anti-PF4 IgG and microparticle (MP) activity were quantified by using commercially available ELISA immunoassays for each of the ESRD (n = 73) and control plasma samples (n = 10). The levels of endogenous glycosaminoglycans (GAGs) were quantified by utilizing a Heparin Red Probe (Redprobes UG, Germany). The collected data were analyzed to demonstrate the relationship between various parameters. All the tested biomarkers were increased in ESRD patients in comparison to healthy controls (p < 0.05). These biomarkers have shown significant correlations within each other except for anti-PF4 Ig G and MPs. The CRP levels were significantly higher in patients who had coronary artery disease (CAD) (p < 0.05), but there was no significant difference in other biomarkers according to the cardiovascular outcomes. In the multivariate analysis, the CRP (odds ratio: 1.19; 95% confidence interval: 1.01-1.41; p: 0.03) value was an independent predictor of CAD. In this study, we demonstrated increased levels of 10 different biomarkers in ESRD patients. The CRP levels can be a good predictor of CAD in ESRD patients.

The Impact of Glycemic Control in Thrombo-Inflammatory Biomarkers in Diabetic Pulmonary Embolism Patients

The Impact of Glycemic Control in Thrombo-inflammatory Biomarkers in Diabetic Pulmonary Embolism Patients. Bulent Kantarcioglu, Alexander O'Hara, Jacob Pozin, Roumika Patil, Maddie Allen, Emily Krupa, Fakiha Siddiqui, Debra Hoppensteadt, Atul Laddu, Amir Darki, Jawed Fareed. Introduction: Venous thromboembolism (VTE) is estimated to affect between 350,000-600,000 individuals annually in the United States. The mortality rate is approximately 100,000 people each year. Diabetes mellitus (DM) is one of the most common metabolic disorders resulting from dysregulated glucose metabolism and is a very important risk factor for cardiovascular disease. Several different factors contribute to thrombotic events in DM; these include inflammatory states, primary hemostatic changes, increased levels of various clotting factors, impaired fibrinolysis, increased oxidative stress, and endothelial dysfunction. However, the results of previous studies were inconsistent to explain the complex relationship between the poor glycemic control in DM and development of PE. HbA1c, which is used as an indicator for glycemic control, may also be an important measurement to quantify the level of thromboinflammatory response that is observed in diabetic patients. Thrombo-inflammatory biomarkers such as D-Dimer, CRP, PAI-1, tPA, TAFI, vWF and endogenous glycosaminoglycans (GAGs) were investigated in several previous studies, however, the relevance of these biomarkers in complex relationships of DM and PE is not completely understood. This study aimed to investigate the role of DM in modulating thromboinflammatory biomarkers in PE patient. Materials & Methods: The diabetic PE patient samples were collected following the ongoing IRB and PERT protocols at Loyola University Medical Center and affiliated hospitals. The levels of thrombo-inflammatory markers (D-Dimer, CRP, PAI-1, tPA, TAFI and vWF) were tested by ELISA methods, endogenous GAGs with heparin red (Red Probes, Muenster, Germany) method. The clinical information of 113 diabetic PE patients, including the HbA1c levels within 3 months of PE diagnosis were collected through chart review of the electronically medical records of the patients. After exclusion of patients with active cancer, circulating levels of each biomarker in diabetic PE patient plasma (n:90) were compared to healthy controls (n:30) and according to HbA1c levels (n:45). Results: The levels of all the tested biomarkers were elevated in diabetic PE patients in comparison to healthy controls (P< 0.05). D-dimer (25.06-fold) and CRP (42.01-fold) have shown the most prominent increases in comparison to healthy controls (Table 1). We did not observe any significant difference in the levels of D-dimer, CRP, PAI-1, t-PA and endogenous GAGs according to HbA1c levels (p< 0.05). The levels of TAFIa (p<0.01) and vWF (p: 0.03) were significantly higher in diabetic patients that had a HbA1c ≥ 7. Conclusion: Our findings support that the increased levels of thromboinflammatory biomarkers and dysregulation of hemostatic pathways are important pathophysiological mechanisms in diabetic PE patients. Increased levels of TAFI and vWF may be the potential contributors of impaired fibrinolysis and endothelial dysregulation in diabetic PE patients who have poor glycemic control. These results underscore the importance of glycemic regulation in diabetic PE patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating the Role of Sulfate Groups for the Binding of Gd3+ Ions to Glycosaminoglycans with NMR Relaxometry.

Glycosaminoglycans (GAGs) are highly negatively charged macromolecules with a large cation binding capacity, but their interaction potential with exogeneous Gd3+ ions is under‐investigated. These might be released from chelates used as Gadolinium‐based contrast agents (GBCAs) for clinical MR imaging due to transmetallation with endogenous cations like Zn2+. Recent studies have quantified how an endogenous GAG sequesters released Gd3+ ions and impacts the thermodynamic and kinetic stability of some GBCAs. In this study, we investigate and compare the chelation ability of two important GAGs (heparin and chondroitin sulfate), as well as the homopolysaccharides dextran and dextran sulfate that are used as models for alternative macromolecular chelators. Our combined approach of MRI‐based relaxometry and isothermal titration calorimetry shows that the chelation process of Gd3+ into GAGs is not just a long‐range electrostatic interaction as proposed for the Manning model, but presumably a site‐specific binding. Furthermore, our results highlight the crucial role of sulfate groups in this process and indicate that the potential of a specific GAG to engage in this mechanism increases with its degree of sulfation. The transchelation of Gd3+ ions from GBCAs to sulfated GAGs should thus be considered as one possible explanation for the observed long‐term deposition of Gd3+ in vivo and related observations of long‐term signal enhancements on T1‐weighted MR images.

Changes in Hemostasis during Liver Transplantation in Children

The objective: to assess changes of clinical and laboratory hemostasic parameters in children at the stages of orthotopic liver transplantation.Subjects and Methods. A single-centre, prospective, pilot study of coagulation test scores was performed in 20 children aged 0 to 11 years with hepatoblastoma after a course of PRETEX IV chemotherapy who underwent liver transplantation from a related donor.Results. Children from 0 to 11 years old with hepatoblastoma, after chemotherapy, retain the normal functional activity of the hemostasis system at the pre-hepatic stage of transplantation: APTT 34.6 (34.45; 38.65) s; prothrombin index (PI) 83.53 (74.09; 87.87) %; INR 1.22 (1.16; 1.42) in the 0‒2 year group, APTT 40.45 (34.9; 41, 68), PI 75.57 (64.41; 80.45) %, and INR 1.39 (1.36; 1.87) in the group aged 3‒11 years. Severe hypocoagulation develops by the end of the hepatic stage and in the first 15 minutes after the start of arterial blood flow through the graft: CT and CFT from 195.0 (189.0; 228.5) s and 163.0 (129.5; 171.5) s to 537.0 (456.0; 1,651.5) s and 468.0 (324.5; 611.5) s (p &lt; 0.05), respectively, in the INTEM test. Hypocoagulation at this time develops due to deficiency of coagulation factors and the release of endogenous glycosaminoglycans from the endothelium transplant, as indicated by an increase in CT and CFT to 666.0 (468.5; 2,209.5) s and 254.5 (203.25; 305.75) s in INTEM, CT to 525.0 (389.0; 2028.0) s with p &lt; 0.05 in HEPTEM in children under the age group of up to 2. Within 1 hour after the start of arterial blood flow, the effects of systemic heparinoid effects persist, which is confirmed by the dynamics of CT 360.0 (219.5; 2,116.5) s with up to 960.0 (560.5; 1,440.5) s with p &lt; 0.05 in the EXTEM test.Conclusion. Changes in hemostasis system parameters during surgery make it possible to judge about rapidly developing coagulation and anticoagulation disorders in children of the presented age groups. Specific changes in the coagulation profile at the stages of liver transplantation in children under the age of 11 inclusive, make it possible to do without routine correction of changes in the hemostasis system.

Endogenous Glycosaminoglycans in Various Pathologic Plasma Samples as Measured by a Fluorescent Quenching Method.

Endogenous glycosaminoglycans (GAGs) with a similar structure to heparin are widely distributed in various tissues. A fluorescence probe, namely Heparin Red, can detect polyanionic GAGs in plasma samples. The purpose of this study is to measure endogenous GAGs in various plasma samples obtained from different pathologic states in comparison to healthy controls utilizing this method. Plasma samples were obtained from patient groups including atrial fibrillation (AF), end-stage-renal-disease (ESRD), diabetes mellitus (DM), sepsis, cancer, liver disease (LD), and pulmonary embolism (PE). Normal human plasma (NHP) was used as healthy controls. The Heparin Red kit from Red Probes (Münster, Germany) was used for the quantification of endogenous GAGs in each sample before and after heparinase I degradation. All results were compiled as group means ± SD for comparison. NHP was found to have relatively low levels of endogenous GAGs with a mean concentration of 0.06 μg/mL. The AF, ESRD, DM, and sepsis patient samples had a mean endogenous GAG concentration of 0.55, 0.72, 0.92, and 0.94 μg/mL, respectively. The levels of endogenous GAGs were highest in cancer, LD, and PE patient plasma samples with a mean concentration of 1.95, 2.78, and 2.83 μg/mL, respectively. Heparinase I degradation resulted in a decline in GAG levels in plasma samples. These results clearly show that detectable Heparin Red sensitive endogenous GAGs are present in circulating plasma at varying levels in various patient groups. Additional studies are necessary to understand this complex pathophysiology.

The Relevance of Anti-PF4 Antibody Isotypes and Endogenous Glycosaminoglycans and their Relationship with Inflammatory Biomarkers in Pulmonary Embolism Patients.

IntroductionPrevious studies have shown that inflammation may contribute to the interplay of endogenous glycosaminoglycans (GAGs) and anti-PF4 antibodies. In this study, we quantified the levels of anti-PF4 antibody isotypes and endogenous GAGs together with inflammatory biomarkers in pulmonary embolism (PE) patients to determine whether there is a relationship in between. Identification of this relationship may provide insight to the complex pathophysiology of PE and HIT and may also be useful for development of potential prognostic, diagnostic and therapeutic interventions.Materials and MethodsPlasma samples from PE patients (n: 210) were analyzed for anti-PF4 antibody isotypes and various thrombo-inflammatory cytokines utilizing commercially available biochip array and ELISA methods. The endogenous GAG levels in PE patients’ plasma were quantified using a fluorescence quenching method. The collected data analyzed to demonstrate the relationship between various parameters.ResultsThe endogenous GAG levels were increased in the PE group (P < .05). The levels of anti-PF4 antibody isotypes were higher in varying levels in comparison to the normal group (P < .05). Inflammatory cytokines have shown varying levels of increase with IL-6, IL-8 and IL-10 showing the most pronounced values. Mortality outcome was related to increased GAGs and some of the cytokines.ConclusionIn this study, we demonstrated increased levels of anti-PF4 antibody isotypes, endogenous GAGs, and inflammatory biomarkers in a large patient cohort in PE. The levels of the endogenous GAGs and inflammatory biomarkers were associated with PE severity and mortality. More studies are needed to understand this complex pathophysiology.

Predictive Role of Blood Cellular Indices and Their Relationship with Endogenous Glycosaminoglycans as Determinants of Inflammatory Biomarkers in Pulmonary Embolism.

IntroductionIn this study, we profiled the levels of blood cellular indices, endogenous glycosaminoglycans (GAGs) and inflammatory biomarkers in a cohort comprised of pulmonary embolism (PE) patients, to determine their inter-relationships. Identification of this relationship may provide insight to the complex pathophysiology of PE and the predictive role of blood cellular indices in acute PE patients.Materials and MethodsPlasma samples from PE patients and healthy controls were analyzed for thrombo-inflammatory biomarkers (IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFN-ɣ, TNF-α, IL-1α, IL-1β, MCP-1, EGF, D-dimer, CRP and MMP-9) using biochip array and ELISA methods. The endogenous GAG levels were quantified using a fluorescence quenching method. The data regarding the blood cellular indices were collected through the review of patient medical records and analyzed to demonstrate their relationship.ResultsThe levels of inflammatory biomarkers and endogenous GAGs were elevated in acute PE patients compared to controls (P < .05). Most of the blood cellular indices have shown significant differences in acute PE patients compared to controls (P < .05). The levels of inflammatory biomarkers, endogenous GAGs and the blood cellular indices have shown significant associations in correlation and multivariable analysis. While NLR, PLR and SII were significantly predicting the 30-day mortality, PNR, ELR and EMR were not sufficient to predict 30-day mortality in acute PE.ConclusionOur results show that the increased thrombo-inflammatory response is associated with the release of GAGs and the changes in blood cellular indices. The predictive role of the blood cellular indices for mortality is dependent on their relationship with the inflammatory response.

Xyloside Derivatives as Molecular Tools to Selectively Inhibit Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis.

Glycosaminoglycan (GAG) side chains of proteoglycans are involved in a wide variety of developmental and pathophysiological functions. Similar to a gene knockout, the ability to inhibit GAG biosynthesis would allow us to examine the function of endogenous GAG chains. However, ubiquitously and irreversibly knocking out all GAG biosynthesis would cause multiple effects, making it difficult to attribute a specific biological role to a specific GAG structure in spatiotemporal manner. Reversible and selective inhibition of GAG biosynthesis would allow us to examine the importance of endogenous GAGs to specific cellular, tissue, or organ systems. In this chapter, we describe the chemical synthesis and biological evaluation of xyloside derivatives as selective inhibitors of heparan sulfate and chondroitin/dermatan sulfate proteoglycan biosynthesis.

Effect of Glycosaminoglycan Replacement on Markers of Interstitial Cystitis In Vitro.

Aims: To examine the effect of three commercial intravesical formulations of glycosaminoglycan on in vitro inflammatory models of IC/BPS to better understand there effect on specific markers of disease. Methods: Human urothelial cells (HTB-4) were cultured under four conditions in the presence or absence of commercial GAG formulations. Cells were cultured under a basal condition or pre-treated with protamine sulfate (100 ng/ml) (damages the endogenous glycosaminoglycan layer), hydrogen peroxide (1%) (a metabolic stressor) or TNFα (10 ng/ml) (creating an inflammatory environment). Each of these four culture conditions was then treated with one of three GAG formulations, CystistatⓇ, iAluRilⓇ and HyacystⓇ. Assays were then performed to examine the effect of the exogenous GAGs on cell viability, cell migration, sGAG production, cytokine and gene expression. Results: All GAG formulations were well tolerated by the HTB-4 cells and supported cell growth and migration. iAluRilⓇ was most effective at stimulating endogenous sGAG production under all conditions, increasing sGAGs by up to 15-fold. All GAG formulations significantly reduced the production of the pro-inflammatory cytokine IL-8 under basal conditions, while no GAG treatment suppressed cytokine production under any other condition. Only CystistatⓇ had a significant effect on HA receptor expression, significantly increasing ICAM-1 expression at 3 h that returned to basal levels at 24 h. No GAG treatment significantly changed the expression of GAG synthesis enzymes (CSGALNACT1, CSGALNACT2) or markers of tissue remodeling (MMP2, TIMP1) and pain (COX-1/PTGS-1, NGF). Conclusions: The data presented in this study reveal that commercial intravesical formulation support cell viability and migration. In addition, the commercial GAG formulations have a mild anti-inflammatory effect in the in vitro model of interstitial cystitis/bladder pain syndrome.

Native Mass Spectrometry Sheds Light on Formation of Deadly Heparin-PF4 Complexes

Native Mass Spectrometry Sheds Light on Formation of Deadly Heparin-PF4 Complexes

Shortening heparan sulfate chains prolongs survival and reduces parenchymal plaques in prion disease caused by mobile, ADAM10-cleaved prions.

Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1+/-) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions.

Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications.

Glycosaminoglycans (GAGs) are polysaccharides ubiquitously found on cell surfaces and in the extracellular matrix (ECM). They regulate numerous cellular signaling events involved in many developmental and pathophysiological processes. GAGs are composed of complex sequences of repeating disaccharide units, each of which can carry many different modifications. The tremendous structural variations account for their ability to bind many proteins and thus, for their numerous functions. Although the sequence of GAG biosynthetic events and the enzymes involved mostly were deduced a decade ago, the emergence of tissue or cell specific GAGs from a nontemplate driven process remains an enigma. Current knowledge favors the hypothesis that macromolecular assemblies of GAG biosynthetic enzymes termed "GAGOSOMEs" coordinate polymerization and fine structural modifications in the Golgi apparatus. Distinct GAG structures arise from the differential channeling of substrates through the Golgi apparatus to various GAGOSOMEs. As GAGs perform multiple regulatory roles, it is of great interest to develop molecular strategies to selectively interfere with GAG biosynthesis for therapeutic applications. In this Account, we assess our present knowledge on GAG biosynthesis, the manipulation of GAG biosynthesis using synthetic xylosides, and the unrealized potential of these xylosides in various biomedical applications. Synthetic xylosides are small molecules consisting of a xylose attached to an aglycone group, and they compete with endogenous proteins for precursors and biosynthetic enzymes to assemble GAGs. This competition reduces endogenous proteoglycan-bound GAGs while increasing xyloside-bound free GAGs, mostly chondroitin sulfate (CS) and less heparan sulfate (HS), resulting in a variety of biological consequences. To date, hundreds of xylosides have been published and the importance of the aglycone group in determining the structure of the primed GAG chains is well established. However, the structure-activity relationship has long been cryptic. Nonetheless, xylosides have been designed to increase HS priming, modified to inhibit endogenous GAG production without priming, and engineered to be more biologically relevant. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. They are small, orally bioavailable, easily excreted, and utilize the host cell biosynthetic machinery to assemble GAGs that are likely nonimmunogenic. Various xylosides have been shown, in different biological systems, to have anticoagulant effects, selectively kill tumor cells, abrogate angiogenic and metastatic pathways, promote angiogenesis and neuronal growth, and affect embryonic development. However, most of these studies utilized the commercially available one or two β-D-xylosides and focused on the impact of endogenous proteoglycan-bound GAG inhibition on biological activity. Nevertheless, the manipulation of cell behavior as a result of stabilizing growth factor signaling with xyloside-primed GAGs is also reckonable but underexplored. Recent advances in the use of molecular modeling and docking simulations to understand the structure-activity relationships of xylosides have opened up the possibility of a more rational aglycone design to achieve a desirable biological outcome through selective priming and inhibitory activities. We envision these advances will encourage more researchers to explore these fascinating xylosides, harness the GAG biosynthetic machinery for a wider range of biomedical applications, and accelerate the successful transition of xyloside-based therapeutics from bench to bedside.

Modulating the Antithrombin-Mediated in Vivo Clearance of Coagulation Factor VIIa

The short half-life of coagulation factor VIIa (FVIIa) in circulation is the result of elimination through multiple pathways of which inactivation by the plasma inhibitor antithrombin (AT) accounts for as much as 65% of the total clearance in humans. Remarkably, the rate of inhibition in vivo is about 30 times greater than the uncatalyzed rate of inhibition in vitro suggesting the presence of rate enhancing components in vivo (Agersø et al. (2011) J Thromb Haemost, 9:333-338). Prime candidates include endogenous heparin-like glycosaminoglycans (GAGs) potentiating the reactivity of antithrombin, or tissue factor (TF) which upon binding to FVIIa increases its susceptibility to inhibition.In the present study site-directed mutagenesis of FVIIa was undertaken to identify variants with altered AT reactivity in order to explore the relationship between the reactivity of FVIIa with AT in vitro and in vivo as well as the nature of endogenous rate enhancing components. The pharmacokinetic properties of FVIIa variants were determined in Sprague Dawley rats as this model recapitulates the aspects of AT-mediated FVIIa clearance observed in humans and allows for interaction of human FVIIa with endogenous rat TF. Similar to the human situation, inactivation of wild-type FVIIa in rat is evident as an accumulation of circulating FVIIa-AT complexes and a progressive divergence of the pharmacokinetic profiles representing FVIIa clot activity and total FVIIa antigen.Initially, the ability to modulate the in vivo complex formation with AT was investigated using two FVIIa variants exhibiting enhanced (>200%) or reduced (<10%) in vitro reactivity with AT, respectively, regardless of the type of cofactor present. Reflecting the in vitro reactivity, clot activity and antigen PK profiles in rats were found to coincide for the AT resistant variant along with essentially no detectable AT complex formation, whereas exacerbated AT complex formation and clot activity:antigen discrepancy was observed for the variant exhibiting enhanced in vitro reactivity.Interestingly, among the generated FVIIa variants with altered AT reactivity, two subsets were identified that displayed differential in vitro reactivity with AT depending on the type cofactor present. Accordingly, one group exhibited a greater susceptibility to inhibition relative to wild-type FVIIa in the presence of heparin but not in the presence of TF, while the other group demonstrated the opposite behavior. Endowed with the ability to report on the cofactor identity from the rate of inhibition relative to wild-type FVIIa, variants from each group were tested for their tendency to accumulate as complexes with AT following intravenous administration to rats. Supporting a contribution from endogenous GAGs to the in vivo inactivation of FVIIa, the measured in vivo peak levels of accumulated FVIIa-AT complexes were found to directly correlate with the in vitro rate constants determined for the variants in the presence of heparin, but not when the cofactor was TF or the combination of TF and heparin.Altogether, these results 1) demonstrate a direct relationship between the in vitro reactivity of FVIIa with AT in the presence of heparin and the clearance of FVIIa through this pathway in vivo, and 2) identify heparin-like GAGs as the likely rate enhancing component of FVIIa inhibition in vivo. DisclosuresØstergaard:Novo Nordisk A/S: Employment. Hansen:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Agersø:Novo Nordisk A/S: Employment. Pedersen:Novo Nordisk A/S: Employment. Glue:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Andersen:Novo Nordisk A/S: Employment. Bjelke:Novo Nordisk A/S: Employment. Breinholt:Novo Nordisk A/S: Employment. Stennicke:Novo Nordisk A/S: Employment. Gandhi:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Hermit:Novo Nordisk A/S: Employment.

Alteration in immune responses toward N-deacetylation of hyaluronic acid.

Hyaluronic acid (HA) is an ubiquitous nonsulfated glycosaminoglycan of the extracellular matrix in all mammalian connective tissues. Along with the age growth, HA will lose its N-acetyl groups in vivo; however, the significance of this physiological process remains largely unknown. Herein, three highly N-deacetylated HAs (dHAs), dHA-5 kDa (Mw: 5 kDa, DD: 100%), dHA-16 kDa (Mw: 16 kDa, DD: 94%) and dHA-110 kDa (Mw: 110 kDa, DD: 72%), were generated after hydrazinolysis. Their capability in the activation of antigen-presenting cells (APCs) was compared with that of their respective HAs. Our results demonstrated that both HAs and dHAs could activate the nuclear factor-kappa B (NF-κB) transcription factor in APCs and induced cytokine production through the Toll-like receptor (TLR)/MyD88 pathway. Notably, the capacity of dHAs in cytokine induction was much lower than that of HAs. In addition, the TLR-2 pathway was much involved following the appearance of zwitterionic motifs in dHAs. Thus, our findings highlight that N-deacetylation renders HA divergences in immune response, which might be implicated in age-induced functional change in endogenous glycosaminoglycans due to the structural modification in vivo.

Glycosaminoglycan Modifications in Duchenne Muscular Dystrophy

Widespread skeletal muscle degeneration and impaired regeneration lead to progressive muscle weakness and premature death in patients with Duchenne muscular dystrophy (DMD). Dystrophic muscles are progressively replaced by nonfunctional tissue because of exhaustion of muscle precursor cells and excessive accumulation of extracellular matrix (ECM). Sulfated glycosaminoglycans (GAGs) are components of the ECM and are increasingly implicated in the regulation of biologic processes, but their possible role in the progression of DMD pathology is not understood. In the present study, we performed immunohistochemical and biochemical analyses of endogenous GAGs in skeletal muscle biopsies of 10 DMD patients and 11 healthy individuals (controls). Immunostaining targeted to specific GAG species showed greater deposition of chondroitin sulfate (CS)/dermatan (DS) sulfate in DMD patient biopsies versus control biopsies. The selective accumulation of CS/DS in DMD biopsies was confirmed by biochemical quantification assay. In addition, high-performance liquid chromatography analysis demonstrated a modification of the sulfation pattern of CS/DS disaccharide units in DMD muscles. In conclusion, our data open up a new path of investigation and suggest that GAGs could represent a new and original therapeutic target for improving the success of gene or cell therapy for the treatment of muscular dystrophies.