Membrane-bound Proteoglycans Research Articles

DUET-01: A first-in-human, phase 1/2 study of BOXR1030 in patients with advanced glypican-3-positive solid tumors.

TPS2681 Background: Glypican-3 (GPC3) is a membrane-bound heparin sulfate proteoglycan involved in cell proliferation that is overexpressed in several tumor types. BOXR1030 is an autologous T-cell therapy that co-expresses a chimeric antigen receptor (CAR) targeting GPC3 on tumor cells and glutamic-oxaloacetic transaminase 2 (GOT2), a mitochondrial enzyme that plays an important role in maintaining mitochondrial function and improving T-cell functionality in the solid tumor microenvironment. In non-clinical studies, BOXR1030 showed GPC3-specific cytotoxicity, proliferation and cytokine release only in the presence of GPC3+ cells. Methods: DUET-01 (NCT05120271) is an open-label, dose escalation and expansion clinical trial to determine a safe dose of BOXR1030 in patients with advanced GPC3-positive solid tumors. Dose escalation will be guided by the occurrence of dose-limiting toxicities (DLTs) within 28 days of dosing. The Bayesian logistic regression model will be used on the accumulated DLT data to estimate the maximum tolerated dose (MTD). The Dose Escalation Committee will select the recommended phase 2 dose (RP2D) to be used in the expansion phase cohort(s) (10-20 subjects each) based on data accumulated in the dose escalation cohorts. Eligible subjects will undergo leukapheresis to obtain T cells for BOXR1030 manufacturing and will receive 3 days of lymphodepleting chemotherapy with fludarabine and cyclophosphamide within 5 days prior to BOXR1030 administration. The starting dose is 0.3 × 106 BOXR1030 T cells/kg body weight. Antitumor activity will be assessed every 6 weeks after cell infusion per Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1 and RECIST for immune-based therapeutics (iRECIST). Six months after BOXR1030 administration, subjects will enter long-term follow-up for up to 15 years. Long-term follow-up assessments will focus on long-term safety and disease status. Main inclusion criteria are histologically confirmed advanced unresectable or metastatic hepatocellular carcinoma, squamous cell carcinoma of the lung, myxoid/round cell liposarcoma, or Merkel cell carcinoma; GPC3 overexpression by immunohistochemistry assay confirmed centrally on tumor specimen taken within 6 months prior to signing consent and after the initiation of the subject’s most recent systemic anticancer therapy; body weight of ≥ 50 kg (≥ 65 kg for dose level 1); and life expectancy > 16 weeks. The primary endpoints are the incidence of DLTs; determination of the MTD and RP2D; the type, frequency and severity of treatment-emergent adverse events; clinically significant abnormal safety laboratory findings and vital signs. Key secondary endpoints include efficacy parameters such as objective response rate, BOXR1030 T-cell expansion and persistence, and levels of inflammatory markers and cytokines. The first patient was treated with BOXR1030 in December 2022. Clinical trial information: NCT05120271 .

Chondroitin sulfate proteoglycan 4: An attractive target for antibody-based immunotherapy.

Multifunctional molecules involved in tumor progression and metastasis have been identified as valuable targets for immunotherapy. Among these, chondroitin sulfate proteoglycan 4 (CSPG4), a significant tumor cell membrane-bound proteoglycan, has emerged as a promising target, especially in light of advances in chimeric antigen receptor (CAR) T-cell therapy. The profound bioactivity of CSPG4 and its role in pivotal processes such as tumor proliferation, migration, and neoangiogenesis underline its therapeutic potential. We reviewed the molecular intricacies of CSPG4, its functional attributes within tumor cells, and the latest clinical-translational advances targeting it. Strategies such as blocking monoclonal antibodies, conjugate therapies, bispecific antibodies, small-molecule inhibitors, CAR T-cell therapies, trispecific killer engagers, and ribonucleic acid vaccines against CSPG4 were assessed. CSPG4 overexpression in diverse tumors and its correlation with adverse prognostic outcomes emphasize its significance in cancer biology. These findings suggest that targeting CSPG4 offers a promising avenue for future cancer therapy, with potential synergistic effects when combined with existing treatments.

The Brain Endothelial Cell Glycocalyx Plays a Crucial Role in the Development of Enlarged Perivascular Spaces in Obesity, Metabolic Syndrome, and Type 2 Diabetes Mellitus.

The brain endothelial cell (BEC) glycocalyx (ecGCx) is a BEC surface coating consisting of a complex interwoven polysaccharide (sweet husk) mesh-like network of membrane-bound proteoglycans, glycoproteins, and glycosaminoglycans (GAGs) covering the apical luminal layer of the brain endothelial cells. The ecGCx may be considered as the first barrier of a tripartite blood-brain barrier (BBB) consisting of (1) ecGCx; (2) BECs; and (3) an extravascular compartment of pericytes, the extracellular matrix, and perivascular astrocytes. Perturbations of this barrier allow for increased permeability in the postcapillary venule that will be permissive to both fluids, solutes, and proinflammatory peripherally derived leukocytes into the perivascular spaces (PVS) which result in enlargement as well as increased neuroinflammation. The ecGCx is known to have multiple functions, which include its physical and charge barrier, mechanical transduction, regulation of vascular permeability, modulation of inflammatory response, and anticoagulation functions. This review discusses each of the listed functions in detail and utilizes multiple transmission electron micrographs and illustrations to allow for a better understanding of the ecGCx structural and functional roles as it relates to enlarged perivascular spaces (EPVS). This is the fifth review of a quintet series that discuss the importance of EPVS from the perspective of the cells of brain barriers. Attenuation and/or loss of the ecGCx results in brain barrier disruption with increased permeability to proinflammatory leukocytes, fluids, and solutes, which accumulate in the postcapillary venule perivascular spaces. This accumulation results in obstruction and results in EPVS with impaired waste removal of the recently recognized glymphatic system. Importantly, EPVS are increasingly being regarded as a marker of cerebrovascular and neurodegenerative pathology.

Endothelial Glycocalyx Injury in SARS-CoV-2 Infection: Molecular Mechanisms and Potential Targeted Therapy.

This review aims at summarizing state-of-the-art knowledge on glycocalyx and SARS-CoV-2. The endothelial glycocalyx is a dynamic grid overlying the surface of the endothelial cell (EC) lumen and consists of membrane-bound proteoglycans and glycoproteins. The role of glycocalyx has been determined in the regulation of EC permeability, adhesion, and coagulation. SARS-CoV-2 is an enveloped, single-stranded RNA virus belonging to β-coronavirus that causes the outbreak and the pandemic of COVID-19. Through the respiratory tract, SARS-CoV-2 enters blood circulation and interacts with ECs possessing angiotensin-converting enzyme 2 (ACE2). Intact glycolyx prevents SARS-CoV-2 invasion of ECs. When the glycocalyx is incomplete, virus spike protein of SARS-CoV-2 binds with ACE2 and enters ECs for replication. In addition, cytokine storm targets glycocalyx, leading to subsequent coagulation disorder. Therefore, it is intriguing to develop a novel treatment for SARS-CoV-2 infection through the maintenance of the integrity of glycocalyx. This review aims to summarize state-of-the-art knowledge of glycocalyx and its potential function in SARS-CoV-2 infection.

Endothelial glycocalyx in retina, hyperglycemia, and diabetic retinopathy.

The endothelial glycocalyx (EG) is a meshlike network present on the apical surface of the endothelium. Membrane-bound proteoglycans, the major backbone molecules of the EG, consist of glycosaminoglycans attached to core proteins. In addition to maintaining the integrity of the endothelial barrier, the EG regulates inflammation and perfusion and acts as a mechanosensor. The loss of the EG can cause endothelial dysfunction and drive the progression of vascular diseases including diabetic retinopathy. Therefore, the EG presents a novel therapeutic target for treatment of vascular complications. In this review article, we provide an overview of the structure and function of the EG in the retina. Our particular focus is on hyperglycemia-induced perturbations in the glycocalyx structure in the retina, potential underlying mechanisms, and clinical trials studying protective treatments against degradation of the EG.

Use of Albumin as a Resuscitative Fluid in the Intensive Care Unit.

Use of Albumin as a Resuscitative Fluid in the Intensive Care Unit.

A new adenine nucleotide transporter located in the ER is essential for maintaining the growth of Toxoplasma gondii.

The lumen of the endoplasmic reticulum (ER) is the subcellular site where secretory protein folding, glycosylation and sulfation of membrane-bound proteins, proteoglycans, and lipids occur. The protein folding and degradation in the lumen of the ER require high levels of energy in the form of ATP. Biochemical and genetic approaches show that ATP must first be translocated across ER membrane by particular transporters before serving as substrates and energy sources in the lumenal reactions. Here we describe an ATP/ADP transporter residing in the ER membranes of T.gondii. Immunofluorescence (IFA) assay in transgenic TgANT1-HA tag revealed that TgANT1 is a protein specifically expressed in the ER. In vitro assays, functional integration of TgANT in the cytoplasmic membrane of intact E. coli cells reveals high specificity for an ATP/ADP antiport. The depletion of TgANT leads to fatal growth defects in T.gondii, including a significant slowdown in replication, no visible plaque formation, and reduced ability to invade. We also found that the amino acid mutations in two domains of TgANT lead to the complete loss of its function. Since these two domains are conserved in multiple species, they may share the same transport mechanism. Our results indicate that TgANT is the only ATP/ADP transporter in the ER of T. gondii, and the lack of ATP in the ER is the cause of the death of T. gondii.

Abstract 1707: Increasing immune cell infiltration in hepatocellular carcinoma tumors using a novel GPC3-targeting aptamer conjugate

Abstract Glypican 3 (GPC3) is a cell membrane-bound heparin sulfate proteoglycan, expressed at the cell surface of many cancer types. It has emerged as a leading target for hepatocellular carcinoma treatment due to its overexpression in aberrant liver cells and low expression in healthy adult tissues. One of the major challenges in treating hepatocellular carcinoma with current immunotherapies is turning cold tumors into immunogenic ones. Unmethylated CpG oligodeoxynucleotides (ODNs) are known immune stimulators, however their use in combination therapy for cancer treatment is limited due to the requirement of intra tumoral injections to prevent systemic toxicity. Here, we describe a unique GPC3-specific aptamer, able to target GPC3-expressing cancer cells in vivo as a means to deliver the immunostimulatory CpG 7909. Systemic administration through intra peritoneal (i.p.) injection of the GPC3 aptamer-CpG conjugate (GRX51) decreases Hep3B tumor volume in a Balb/c nude mouse xenograft model by ~30-50% depending on treatment schedule, while also increasing immune cell infiltration. Final tumor volumes of GRX51-treated animals are consistent with tumor volumes of animals treated with the standard of care compound, sorafenib tosylate. Due to its GPC3-targeting capacity, systemic dosing of GRX51 (15 mg/kg) is less toxic than systemic administration with equimolar amounts of CpG 7909 alone (5 mg/kg), with a comparable effect on tumor shrinkage. While both GRX51 and CpG 7909 induce immune cell recruitment, GRX51 treated mice had a final immune:tumor cell ratio of 2.45 ± 0.56, while CpG 7909-treated animals had a ratio of 1.51 ± 0.95 relative to the vehicle control after a 7-day on/7-day off daily dosing schedule. Using the Hep3B xenograft in CD34+ humanized mice, we also showed GRX51 primes tumors for combination treatments with FDA approved checkpoint inhibitors. Mice were treated with GRX51 (7.5 mg/kg daily i.p. dose, 11 days), the PD-1R inhibitor pembrolizumab (10 mg/kg once every 3 days i.p., 11 days), or a combination of the two drugs. The GRX51 treatment alone outperformed pembrolizumab alone, reducing tumor size by 33% and 18% respectively. Notably, the combination of GRX51 with pembrolizumab reduced tumor size by 63%. Together, our data show that GRX51 is a novel GPC3-targeting molecule capable of reducing tumor size and priming GPC3-positive tumors for combination immunotherapy, without inducing major systemic toxicity with i.p. injection. Citation Format: Christina McGuire, Jin Yuan, Aaron Ball, Justin Sonberg, Yuxun Wang, Kristin Thompson, Shuhao Zhu. Increasing immune cell infiltration in hepatocellular carcinoma tumors using a novel GPC3-targeting aptamer conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1707.

Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation.

Basal-like breast cancer is characterized by an aggressive clinical outcome and presence of metastasis, for which effective therapies are unavailable. We have previously shown that chondroitin 4-O-sulfotransferase-1 (C4ST-1) controls the invasive properties of the basal-like breast cancer cell line BT-549 by inducing matrix metalloproteinase (MMP) expression through the N-cadherin/β-catenin pathway. Here we report that C4ST-1 controls the proliferation of BT-549 cells via the MMP-dependent cleavage of syndecan-1. Syndecan-1 is a membrane-bound proteoglycan associated with an aggressive phenotype and poor prognosis in breast cancer. In addition, the cleavage of syndecan-1 at a specific juxtamembrane cleavage site is implicated in the pathophysiological response in breast cancer. Knockout of C4ST-1 remarkably suppressed both the cleavage of syndecan-1 and proliferation of BT-549 cells. Kinases (AKT1, ERK1/2, PI3K, and STAT3) comprising cancer proliferative pathways are phosphorylated in C4ST-1 knockout cells at a level similar to that in parental BT-549 cells, whereas levels of phosphorylated S6 kinase and SUMOylated AKT (hyperactivated AKT observed in breast cancer) decreased in C4ST-1 knockout cells. An MMP inhibitor, GM6001, suppressed the small ubiquitin-like modifier (SUMO) modification of AKT, suggesting that cleavage of syndecan-1 by MMPs is involved in the SUMO modification of AKT. Forced expression of the cytoplasmic domain of syndecan-1, which is generated by MMP-dependent cleavage, increased the SUMO modification of AKT and global protein SUMOylation. Furthermore, syndecan-1 C-terminal domain-expressing BT-549 cells were more proliferative and sensitive to a potent SUMOylation inhibitor, tannic acid, compared with BT-549 cells transfected with an empty expression vector. These findings assign new functions to the C-terminal fragment of syndecan-1 generated by MMP-dependent proteolysis, thereby broadening our understanding of their physiological importance and implying that the therapeutic inhibition of syndecan-1 cleavage could affect the progression of basal-like breast cancer.

The significance of glycocalyx in medicine

Introduction. The glycocalyx is a gel-like layer covering the membrane of many cells, especially cells of epithelial tissue. It consists of membrane-bound proteoglycans, glycosaminoglycan chains, glycoproteins, and adjacent proteins. Glycocalyx is necessary in maintaining the permeability of vessels, modulation of inflammatory responses and interactions between cells. It is also involved in cell adherence, mobility, mechanotransduction, regulation of the cell cycle and cell. Abnormalities in the structure and function of the glycocalyx underlie many diseases and disorders such as dry eyes disease, diabetes and its complications as well as sepsis. Aim. In this review, we present the current view on the role of glycocalyx in human diseases. Material and methods. This review was performed according to latest literature from the following databases: EBSCO, PubMed, Science Direct, and Springer Link. Analysis of the literature. Pathological mechanisms such as disruption of the glycocalyx barrier and decreased hydration of the ocular epithelial surface cause dry eye disease. During hyperglycaemia, glycocalyx dysfunction occurs, which leads to its dysfunction and activation of the prothrombotic system. Moreover, the increase in the concentration of hyaluronidase leads to increase in the plasma hyaluronan levels and promotion of endothelial dysfunction. Additionally, degradation of glycocalyx in sepsis prevails over increased synthesis of its components strongly favors its enhanced enzymatic degradation. Conclusion. A better understanding of glycocalyx impairment in disease could alter therapeutic strategies to improve patient outcomes.

Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC–MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

Adhesion of freshwater sponge cells mediated by carbohydrate-carbohydrate interactions requires low environmental calcium.

Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and β-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters.

253. Sublingual microvascular density and glycocalyx barrier dynamics, during and after normal and preeclamptic pregnancy

Introduction The apical surface of the vascular endothelium comprises a complex meshwork of membrane-bound proteoglycans and glycoproteins, the glycocalyx. The glycocalyx has important vasoregulatory roles; e.g., growth factor binding, signal transduction, coagulation and permeability. Glycocalyx damage has been implicated in several vascular diseases. Hypothesis Glycocalyx structural barrier integrity and microvascular perfusion are reduced in women with preeclampsia (PE). Methods We evaluated 17 women with PE and 27 with uncomplicated pregnancy (NL), just prior to delivery (LD) and at 24–48 h postpartum (PP). The sublingual microvasculature was interrogated using sidestream-darkfield imaging to compare glycocalyx integrity, microvascular density and percentage of perfused vascular segments. Glycocalyx integrity (for vessels 5–25 μm) was measured as depth of the RBC accessible region(AR), in which greater AR signifies diminished ability of glycocalyx to exclude RBCs. Plasma soluble syndecan-1, a shed glycocalyx component, was measured by ELISA. Results Microvascular density (segments/mm2) was greater in NL-LD (478 ± 28) compared to PE-LD (378 ± 35 p = 0.002), decreasing in both NL-PP (355 ± 28) and PE-PP (306 ± 35). Perfusion was not different between groups. AR in NL-LD (2.33 ± 0.05microns) was surprisingly greater than PE-LD (2.17 ± 0.06 p = 0.03), or NL-PP (2.19 ± 0.05 p = 0.05) but not different from PE-PP (2.26 ± 0.06). The change in AR between timepoints (LD-PP) indicates postpartum improvement in barrier function in NL (but not PE) group (−0.09 ± 0.4 vs 0.08 ± 0.3 p = 0.05). Maternal plasma syndecan-1 was higher in NL-LD (1126 ± 110.3 ng/mL) compared to PE-LD (715.5 ± 112.1 ng/mL; p = 0.008), and declined in both groups PP (NL-PP 210 ± 98, PE-PP 173 ± 120; NS). Conclusion Women with PE exhibited a reduction in functional microvascular density compared to NL. The increase in RBC accessibility(AR) in NL compared to PE was opposite to our hypothesis. This reduction in endothelial glycocalyx barrier function was accompanied by a higher level of circulating syndecan-1 prior to delivery in NL. The physiologic significance of these glycocalyx changes remain to be determined. Funding PO1-HD-30367, R21-HD-83659, AHA16SFRN27810001

209. The microvascular endothelial glycocalyx: impaired barrier function in preeclampsia with small gestational age neonates

Introduction The glycocalyx, the endothelial lining of membrane-bound proteoglycans and glycoproteins, serves as a functional barrier between flowing red blood cells (RBC) and the endothelial cell membrane. Recent studies suggest that damage to the glycocalyx, which can be reflected by deeper radial penetration of RBC into the glycocalyx, contributes to microvascular dysfunction. Hypothesis We hypothesized that women with preeclampsia, especially those who deliver a small-for-gestational age neonate (PE + SGA, birthweight Methods We compared 30 women with a normotensive pregnancy (NP) to 30 women with preeclampsia (PE), matched for gestational age at time of analysis and presence/absence of labor. Using sidestream dark field imaging of the sublingual microcirculation, we measured 1) the accessibility of the RBC into the glycocalyx layer (perfused boundary region, PBR), defined as the difference between median and maximal RBC column widths, and 2) the microvascular density (total length of perfused microvessels/mm2 area). In healthy vessels, PBR is relatively small due to limited penetration of RBC into the glycocalyx. Results We observed no significant group differences in microvascular density (229 ± 75 mm/mm2 PE, 247 ± 88 mm/mm2 NP, P = 0.48) or PBR (2.20 ± 0.22 μm PE, 2.14 ± 0.30 μm NP, P = 0.14). However, the subset of women with PE + SGA (n = 12) evidenced a significant reduction in vascular density (205 ± 67 vs 306 ± 71 mm/mm2, P Discussion The increase in RBC encroachment (PBR) into the microvascular glycocalyx in PE + SGA compared to NP indicates a change in glycocalyx structure which may be associated with a reduced vascular protective function of the glycocalyx, possibly contributing to endothelial and microvascular dysfunction. Funded by NIH-NICHD (P01 HD030367, R21 HD083659), and AHA16SFRN27870000.

Protocol for a pilot randomized controlled trial comparing plasma with balanced crystalloid resuscitation in surgical and trauma patients with septic shock

BackgroundSeptic shock is a public health problem with high mortality. There remains a knowledge gap regarding the optimal resuscitation fluid to improve clinical outcomes, and the underlying mechanism by which...

Endothelial Progenitor Cells: New Targets for Therapeutics for Inflammatory Conditions With High Cardiovascular Risk.

Over the past decade, we have witnessed an exponential growth of interest into the role of endothelial progenitor cells (EPCs) in cardiovascular disease. While the major thinking revolves around EPC angiogenic repair properties, we have used a hypothesis-driven approach to discover disease-related defects in their characteristics and based on these findings, have identified opportunities for functional enhancement, which offer an exciting avenue for translation into clinical intervention. In this review, we focus on two groups; circulating myeloid angiogenic cells (MACs) and late outgrowth endothelial colony forming cells (ECFCs), and will discuss the unique properties and defects of each population, as new insights have been gained into the potential function of each sub-type using current techniques and multiomic technology. We will discuss their role in inflammatory disorders and alterations in mitochondrial function. In addition, we share key insights into the glycocalyx, and propose this network of membrane-bound proteoglycans and glycoproteins, covering the endothelium warrants further investigation in order to clarify its significance in ECFC regulation of vascularization and angiogenesis and ultimately for potential translational therapeutic aspects.

OS 17-09 URIC ACID-INDUCED ENDOTHELIAL-TO-MESENCHYMAL TRANSITION (Endo-MT) VIA OXIDATIVE STRESS AND GLYCOCALYX SHEDDING

Objective: Recent data suggested a role of uric acid (UA) in the pathogenesis of cardiovascular and renal disease. Endothelial dysfunction, which is characterized by a decrease in nitric oxide, is regarded as the key mechanism of UA-induced vascular disease. Endo-MT is an early process of endothelial dysfunction, and is also known to play a role in the progression of renal fibrosis. Glycocalyx is a structure covering endothelium composed of membrane-bound proteoglycans and glycoproteins associated with adsorbed plasma components, which may lead to endothelial dysfunction via intraluminal shedding. Design and Method: Endo-MT was evaluated by cell morphology and the expression of the endothelial markers, VE-cadherin or CD31 and the mesenchymal marker, α-SMA by real time PCR, western blotting (WB) and immunocytochemistry in HUVECs and animal model of hyperuricemia (Sprague-Dawley rats fed with 2% oxonic acid for 6 weeks). NAPDH oxidase (NOX) activity, reactive oxygen species (ROS) production, endothelial permeability and glycocalyx shedding were evaluated by WB and ELISA. Results: Stimulation of HUVEC with UA resulted in an alteration of cell morphology into fibroblastoid cells associated with a decrease in CD31 and VE-cadherin and de-novo α-SMA expression from 24 hours. UA increased ROS production via NOX (15 min) and mitochondrial activation (6 hours) with an increase in glycocalyx shedding (6 hours), which were blocked by an inhibitor of organic anion transporter, probenecid (5 μM). Anti-oxidant treatment [NAC (5mM), apocynin (100 μM), and mitotempo (10 μM)] ameliorated endo-MT and glycocalyx shedding in HUVEC. Matrix metalloproteinase inhibitor, GM6001, also alleviated UA-induced endoMT. In the kidney of hyperuricemic rats, endothelial staining in peritubular capillaries was decreased with de-novo staining of α-SMA, which was ameliorated allopurinol treatment. Conclusions: UA per se induced a phenotypic transition of endothelial cells via oxidative stress and glycocalyx shedding, which could be one of the mechanisms of UA-induced vascular disease.

Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability.

Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease.

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans

Early metazoans had to evolve the first cell adhesion mechanism addressed to maintain a distinctive multicellular morphology. As the oldest extant animals, sponges are good candidates for possessing remnants of the molecules responsible for this crucial evolutionary innovation. Cell adhesion in sponges is mediated by the calcium-dependent multivalent self-interactions of sulfated polysaccharides components of extracellular membrane-bound proteoglycans, namely aggregation factors. Here, we used atomic force microscopy to demonstrate that the aggregation factor of the sponge Desmapsamma anchorata has a circular supramolecular structure and that it thus belongs to the spongican family. Its sulfated polysaccharide units, which were characterized via nuclear magnetic resonance analysis, consist preponderantly of a central backbone composed of 3-α-Glc1 units partially sulfated at 2- and 4-positions and branches of Pyr(4,6)α-Gal1→3-α-Fuc2(SO3)1→3-α-Glc4(SO3)1→3-α-Glc→4-linked to the central α-Glc units. Single-molecule force measurements of self-binding forces of this sulfated polysaccharide and their chemically desulfated and carboxyl-reduced derivatives revealed that the sulfate epitopes and extracellular calcium are essential for providing the strength and stability necessary to sustain cell adhesion in sponges. We further discuss these findings within the framework of the role of molecular structures in the early evolution of metazoans.

Endothelial pathophysiology, glycosaminoglycans and glycocalyx

The endothelial glycocalyx can be described as a network of membrane-bound proteoglycans and glycoproteins, covering the luminal surface of endothelial cells. Over the past decades, structural properties and functions of glycocalyx have been increasingly examined, underlining its role in many physiological processes. This article provides the basis on composition and functions of the endothelial glycocalyx. The Author also describes the so called “endothelial dysfunction” and how it can lead to the development of pathological inflammatory processes and consequent vascular diseases.