Glycocalyx Degradation Research Articles

PC-FACS

PC-FACS

Metastatic cancer cell attachment to endothelium is promoted by endothelial glycocalyx sialic acid degradation.

While it is known that cancer cell interactions with vascular endothelial cells (ECs) drive metastatic cancer cell extravasation from blood vessels into secondary tumor sites, the mechanisms of action are still poorly understood. Here, we tested the hypothesis that neuraminidase‐induced degradation of EC surface glycocalyx (GCX), particularly the sialic acid (SA) residue components of the GCX, will substantially increase metastatic cancer cell attachment to ECs. To our knowledge, our study is the first to isolate the role of GCX SA residues in cancer cell attachment to the endothelium, which were found to be differentially affected by the presence of neuraminidase and to indeed regulate metastatic cancer cell homing to ECs. We hope that this work will eventually translate to identification of EC GCX‐based cancer markers that can be therapeutically targeted to hinder the progression of metastasis.

Is microcirculatory assessment ready for regular use in clinical practice?

The present review discusses the current role of microcirculatory assessment in the hemodynamic monitoring of critically ill patients. Videomicroscopic techniques have demonstrated that microvascular perfusion is altered in critically ill patients, and especially in sepsis. These alterations are associated with organ dysfunction and poor outcome. Handheld microscopes can easily be applied on the sublingual area of critically ill patients. Among the specific limitations of these techniques, the most important is that these can mostly investigate the sublingual microcirculation. The representativity of the sublingual area may be questioned, especially as some areas may sometimes be more affected than the sublingual area. Also, evaluation of the sublingual area may be difficult in nonintubated hypoxemic patients. Alternative techniques include vasoreactivity tests using either transient occlusion or performing a thermal challenge. These techniques evaluate the maximal dilatory properties of the microcirculation but do not really evaluate the actual microvascular perfusion. Focusing on the glycocalyx may be another option, especially with biomarkers of glycocalyx degradation and shedding. Evaluation of the glycocalyx is still largely experimental, with different tools still in investigation and lack of therapeutic target. Venoarterial differences in PCO2 are inversely related with microvascular perfusion, and can thus be used as surrogate for microcirculation assessment. Several limitations prevent the regular use in clinical practice. The first is the difficult use of some of these techniques outside research teams, whereas nurse-driven measurements are probably desired. The second important limitation for daily practice use is the lack of uniformly defined endpoint. The final limitation is that therapeutic interventions affecting the microcirculation are not straightforward. Clinical and biological surrogates of microcirculatory assessment can be used at bedside. The role of microvideoscopic techniques is still hampered by the lack of clearly defined targets as well as interventions specifically targeting the microcirculation.

Berberine inhibits low shear stress-induced glycocalyx degradation via modulating AMPK and p47phox/Hyal2 signal pathway

A characteristic of endothelia damage and repair is the turnover of extracellular matrix components. As a part of extracellular matrix glycosaminoglycan (GAG), hyaluronic acid (HA, main component of glycocalyx) is not only involved in inflammation, proliferation, differentiation of cells, and tissue remodeling, but also functions as a barrier of endothelium via preventing blood flow-induced injury from endothelial layer. Therefore, the metabolism of hyaluronic acid could allow the fine-tuning of cell behavior. In this study, we found that low shear stress decreased the expression of hyaluronic acid, whereas pretreatment with berberine could significantly increase the expression of hyaluronic acid in vitro and in vivo. On this background, it is very important to better understand the beneficial effect of berberine (BBR) on low shear stress-induced degradation of hyaluronic acid and its potential mechanism. By using siRNA and inhibitors, we testified that AMP-activated protein kinase (AMPK) and p47phox/hyaluronidase 2 (Hyal2) signaling pathway involved in the modulation of hyaluronic acid metabolism. Further, berberine, by increasing AMPK phosphorylation, decreased the dissociation of p47phox/Hyal2, and subsequently inhibited Hyal2 activation and p47phox phosphorylation, leading to the metabolic maintaining of hyaluronic acid. Importantly, we primarily demonstrated a direct binding between AMPKα and p47phox in HUVECs by co-immunoprecipitation. On the other hand, berberine also increased the expression of hyaluronic acid synthase 2 (HAS2) by regulating AMPKα/p47phox signaling pathway. Taken together, berberine treatment can attenuate low shear stress-induced hyaluronic acid degradation via increasing phosphorylation of AMPKa, and then not only downregulates p47phox and Hyal2 activity but also upregulates the expression of HAS2.

Circulating heparin oligosaccharides rapidly target the hippocampus in sepsis, potentially impacting cognitive functions

Sepsis induces heparanase-mediated degradation of the endothelial glycocalyx, a heparan sulfate-enriched endovascular layer critical to vascular homeostasis, releasing highly sulfated domains of heparan sulfate into the circulation. These domains are oligosaccharides rich in heparin-like trisulfated disaccharide repeating units. Using a chemoenzymatic approach, an undecasaccharide containing a uniformly 13C-labeled internal 2-sulfoiduronic acid residue was synthesized on a p-nitrophenylglucuronide acceptor. Selective periodate cleavage afforded a heparin nonasaccharide having a natural structure. This 13C-labeled nonasaccharide was intravenously administered to septic (induced by cecal ligation and puncture, a model of polymicrobial peritonitis-induced sepsis) and nonseptic (sham) mice. Selected tissues and biological fluids from the mice were harvested at various time points over 4 hours, and the 13C-labeled nonasaccharide was recovered and digested with heparin lyases. The resulting 13C-labeled trisulfated disaccharide was quantified, without interference from endogenous mouse heparan sulfate/heparin, using liquid chromatography-mass spectrometry with sensitive and selective multiple reaction monitoring. The 13C-labeled heparin nonasaccharide appeared immediately in the blood and was rapidly cleared through the urine. Plasma nonasaccharide clearance was only slightly prolonged in septic mice (t1/2 ∼ 90 minutes). In septic mice, the nonasaccharide penetrated into the hippocampus but not the cortex of the brain; no hippocampal or cortical brain penetration occurred in sham mice. The results of this study suggest that circulating heparan sulfates are rapidly cleared from the plasma during sepsis and selectively penetrate the hippocampus, where they may have functional consequences.

Sphingosine‐1‐phosphate Enhances Mitochondrial Integrity and Endothelial Barrier Function Following Hemorrhagic Shock

Excessive microvascular permeability is a serious complication involved in traumatic injury and inflammatory diseases. Sphingosine‐1‐phosphate (S1P) has known endothelial barrier‐protective properties, and has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and hemorrhagic shock and resuscitation (HSR). However, the mechanisms of S1P‐induced barrier protection during HSR are unknown. In the current study, we tested the hypothesis that S1P could enhance endothelial barrier function during hemorrhagic shock by preserving the integrity of junction proteins and the endothelial glycocalyx, and protecting mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Assessment of adherens junction protein localization was done by immunofluorescence confocal microscopy after HSR. Cultured rat intestinal microvascular endothelial cell (RIMEC) monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. Our in vivo results show that S1P protects against HSR‐induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. Besides that, S1P protects RIMECs against mitochondrial dysfunction‐induced endothelial barrier dysfunction and glycocalyx degradation by blocking complex III inhibition. Our results show that S1P protects against HSR‐induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity, preventing excess microvascular permeability.Support or Funding InformationSupported by NIH grants R01HL098215, R01GM120774, R01HL070752 and R01GM097270.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The targeted inhibition of neuraminidase reverses endothelial glycocalyx degradation and improves endothelial function in type 2 diabetes

Endothelial dysfunction is a hallmark vascular abnormality of type 2 diabetes (T2D) that is characterized by blunted vasodilatory responses to physiological stimuli such as blood flow‐induced shear stress. Previously, we have reported that plasma neuraminidase levels are increased in a mouse model of diet‐induced obesity that is insulin resistant and considered pre‐diabetic. Neuraminidase is an enzyme that degrades the glycocalyx, which is a structure essential for the mechanotransduction of shear stress in the process of flow‐mediated dilation (FMD). Therefore, we hypothesized that circulating neuraminidase is implicated in the degradation of the endothelial glycocalyx and the diminished FMD that are associated with T2D, and that inhibition of circulating neuraminidase activity is an effective means to restore glycocalyx integrity and endothelial function in diabetic mice. In support of this hypothesis, we provide evidence that endothelial function, as assessed by FMD and insulin‐stimulated vasodilation, is blunted in T2D vs. non‐T2D human subjects and that this impairment concurs with elevated plasma neuraminidase activity and increased concentrations of its enzymatic byproduct, sialic acid. We further demonstrate that ex vivo exposure of mouse arteries to neuraminidase significantly reduces FMD. In cultured endothelial cells, we found that neuraminidase sheds sialic acid and Syndecan‐1, which are components of the glycocalyx and that the neuraminidase inhibitor, Zanamivir, attenuates this shedding. Moreover, in an animal model of diabetes (db/db mice), we demonstrate that Zanamivir reduces plasma neuraminidase activity and concomitantly improves femoral artery FMD and vasodilatory responses to acetylcholine and insulin. We also show that endothelial stiffness, assessed with atomic force microscopy using en face aortic samples, is significantly reduced in Zanamivir‐treated compared to placebo‐treated db/db mice. We conclude that inhibition of plasma neuraminidase activity represents a novel therapeutic approach to ameliorate endothelial dysfunction and cardiovascular risk in T2D patients by reversing glycocalyx degradation.Support or Funding InformationNational Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Circulating heparan sulfate fragments mediate septic cognitive dysfunction.

Septic patients frequently develop cognitive impairment that persists beyond hospital discharge. The impact of sepsis on electrophysiological and molecular determinants of learning is underexplored. We observed that mice that survived sepsis or endotoxemia experienced loss of hippocampal long-term potentiation (LTP), a brain-derived neurotrophic factor-mediated (BDNF-mediated) process responsible for spatial memory formation. Memory impairment occurred despite preserved hippocampal BDNF content and could be reversed by stimulation of BDNF signaling, suggesting the presence of a local BDNF inhibitor. Sepsis is associated with degradation of the endothelial glycocalyx, releasing heparan sulfate fragments (of sufficient size and sulfation to bind BDNF) into the circulation. Heparan sulfate fragments penetrated the hippocampal blood-brain barrier during sepsis and inhibited BDNF-mediated LTP. Glycoarray approaches demonstrated that the avidity of heparan sulfate for BDNF increased with sulfation at the 2-O position of iduronic acid and the N position of glucosamine. Circulating heparan sulfate in endotoxemic mice and septic humans was enriched in 2-O- and N-sulfated disaccharides; furthermore, the presence of these sulfation patterns in the plasma of septic patients at intensive care unit (ICU) admission predicted persistent cognitive impairment 14 days after ICU discharge or at hospital discharge. Our findings indicate that circulating 2-O- and N-sulfated heparan sulfate fragments contribute to septic cognitive impairment.

Degradation of Glycocalyx and Multiple Manifestations of Endothelial Dysfunction Coincide in the Early Phase of Endothelial Dysfunction Before Atherosclerotic Plaque Development in Apolipoprotein E/Low-Density Lipoprotein Receptor-Deficient Mice.

BackgroundThe impairment of endothelium‐dependent vasodilation, increased endothelial permeability, and glycocalyx degradation are all important pathophysiological components of endothelial dysfunction. However, it is still not clear whether in atherosclerosis, glycocalyx injury precedes other features of endothelial dysfunction or these events coincide.Methods and ResultsHerein, we demonstrate that in 4‐ to 8‐week‐old apolipoprotein E/low‐density lipoprotein receptor‐deficient mice, at the stage before development of atherosclerotic plaques, impaired acetylcholine‐induced vasodilation, reduced NO production in aorta, and increased endothelial permeability were all observed; however, flow‐mediated dilation in the femoral artery was fully preserved. In 4‐week‐old mice, glycocalyx coverage was reduced and endothelial stiffness was increased, whereas glycocalyx length was significantly decreased at 8 weeks of age. Early changes in endothelial function were also featured by increased plasma concentration of biomarkers of glycocalyx disruption (endocan), biomarkers of endothelial inflammation (soluble vascular cell adhesion molecule 1), increased vascular permeability (angiopoietin 2), and alterations in hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). In 28‐week‐old mice, at the stage of advanced atherosclerotic plaque development, impaired NO production and nearly all other features of endothelial dysfunction were changed to a similar extent, compared with the preatherosclerotic plaque phase. The exceptions were the occurrence of acetylcholine‐induced vasoconstriction in the aorta and brachiocephalic artery, impaired flow‐mediated vasodilation in the femoral artery, and further reduction of glycocalyx length and coverage with a concomitant further increase in endothelial permeability.ConclusionsIn conclusion, even at the early stage before the development of atherosclerotic plaques, endothelial dysfunction is a complex multifactorial response that has not been previously appreciated.

AMP-activated protein kinase regulates glycocalyx impairment and macrophage recruitment in response to low shear stress.

Low shear stress (LSS) increases degradation of the endothelial glycocalyx, leading to production of endothelial inflammation and atherosclerosis. However, the underlying mechanisms of how LSS diminishes the endothelial glycocalyx remain unclear. We showed that LSS inactivated AMPK, enhanced Na+-H+ exchanger (NHE)1 activity, and induced glycocalyx degradation. Activation of AMPK prevented LSS-induced NHE1 activity and endothelial glycocalyx impairment. We further identified hyaluronidase 2 (HYAL2) as a mediator of endothelial glycocalyx impairment in HUVECs exposed to LSS. Inactivation of AMPK by LSS up-regulates the activity of HYAL2, which acts downstream of NHE1. We characterized a left common carotid artery partial ligation (PL) model of LSS in C57BL/6 mice. The results showed decreased expression of hyaluronan (HA) in the endothelial glycocalyx and decreased thickness of the endothelial glycocalyx in PL mice. Pharmacological activation of AMPK by ampkinone not only attenuated glycocalyx impairment due to HA degradation but also blocked vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression increase and macrophage recruitment in the endothelia of PL mice. Our results revealed that AMPK dephosphorylation induced by LSS activates NHE1 and HYAL2 to promote HA degradation and glycocalyx injury, which may contribute to endothelial inflammatory reaction and macrophage recruitment.-Zhang, J., Kong, X., Wang, Z., Gao, X., Ge, Z., Gu, Y., Ye, P., Chao, Y., Zhu, L., Li, X., Chen, S. AMP-activated protein kinase regulates glycocalyx impairment and macrophage recruitment in response to low shear stress.

Correlation between acute degradation of the endothelial glycocalyx and microcirculation dysfunction during cardiopulmonary bypass in cardiac surgery

ObjectiveThe association between the shedding of the endothelial glycocalyx (EG) and the pathogenesis of microcirculatory perfusion disturbances has been discussed in experimental studies. This discussion, however, has limited relevance in a clinical setting. We investigated EG shedding in patients undergoing cardiopulmonary bypass (CPB) and its association with alterations in microvascular perfusion. MethodsThe plasma levels of syndecan-1, heparan sulfate, and hyaluronan were used as markers of glycocalyx degradation. Microcirculatory parameters included perfused vessel density (PVD) and De Backer Scores. Sidestream dark field imaging (SDF) was applied to visualize sublingual microcirculation during the preoperative resting state (T0), after sternum splitting, after aortic clamping, 5 min before aortal declamping, 1 h after CPB (T4), 4 h after CPB, 24 h after CPB (T6), and 48 h after CPB. ResultsThirty patients undergoing cardiac surgery were recruited. The plasma levels of glycocalyx degradation markers increased after CPB. This increase indicated severe glycocalyx shedding at T4 relative to that at T0. By T6, the plasma levels of glycocalyx degradation markers had decreased to baseline levels in a stepwise manner. PVD and the De Backer Scores decreased at T4 and recovered at T6. Glycocalyx marker concentrations were correlated with microvascular alterations during cardiac surgery. ConclusionsGlycocalyx components are closely related to microcirculation perfusion disorders. Damage to the glycocalyx during surgery with CPB may play a key role in microcirculation perfusion dysfunction.

Role of deformable cancer cells on wall shear stress-associated-VEGF secretion by endothelium in microvasculature

Endothelial surface layer (glycocalyx) is the major physiological regulator of tumor cell adhesion to endothelium. Cancer cells express vascular endothelial growth factor (VEGF) which increases the permeability of a microvessel wall by degrading glycocalyx. Endothelial cells lining large arteries have also been reported, in vitro and in vivo, to mediate VEGF expression significantly under exposure to high wall shear stress (WSS) > 0.6 Pa. The objective of the present study is to explore whether local hemodynamic conditions in the vicinity of a migrating deformable cancer cell can influence the function of endothelial cells to express VEGF within the microvasculature. A three-dimensional model of deformable cancer cells (DCCs) migrating within a capillary is developed by applying a massively parallel hemodynamics application to simulate the fluid-structure interaction between the DCC and fluid surrounding the DCC. We study how dynamic interactions between the DCC and its local microenvironment affect WSS exposed on endothelium, under physiological conditions of capillaries with different diameters and flow conditions. Moreover, we quantify the area of endothelium affected by the DCC. Our results show that the DCC alters local hemodynamics in its vicinity up to an area as large as 40 times the cancer cell lateral surface. In this area, endothelium experiences high WSS values in the range of 0.6–12 Pa. Endothelial cells exposed to this range of WSS have been reported to express VEGF. Furthermore, we demonstrate that stiffer cancer cells expose higher WSS on the endothelium. A strong impact of cell stiffness on its local microenvironment is observed in capillaries with diameters <16 μm. WSS-induced-VEGF by endothelium represents an important potential mechanism for cancer cell adhesion and metastasis in the microvasculature. This work serves as an important first step in understanding the mechanisms driving VEGF-expression by endothelium and identifying the underlying mechanisms of glycocalyx degradation by endothelium in microvasculature. The identification of angiogenesis factors involved in early stages of cancer cell-endothelium interactions and understanding their regulation will help, first to develop anti-angiogenic strategies applied to diagnostic studies and therapeutic interventions, second to predict accurately where different cancer cell types most likely adhere in microvasculature, and third to establish accurate criteria predisposing the cancer metastasis.

Early Onset Preeclampsia Is Associated With Glycocalyx Degradation and Reduced Microvascular Perfusion.

BackgroundThe endothelial glycocalyx is a vasoprotective barrier between the blood and endothelium. We hypothesized that glycocalyx degradation is present in preeclampsia, a pregnancy‐specific hypertensive disorder characterized by endothelial dysfunction and activation.Methods and ResultsWe examined the sublingual glycocalyx noninvasively using sidestream dark field imaging in the third trimester among women with normotensive pregnancies (n=73), early (n=14) or late (n=29) onset preeclampsia, or gestational diabetes mellitus (n=21). We calculated the width of the glycocalyx that was permeable to red blood cells (called the perfused boundary region, a measure of glycocalyx degradation) and the percentage of vessels that were filled with red blood cells ≥50% of the time (a measure of microvascular perfusion). In addition, we measured circulating levels of glycocalyx components, including heparan sulfate proteoglycans, hyaluronic acid, and SDC1 (syndecan 1), in a subset of participants by ELISA. Repeated‐measures ANOVA was performed to adjust for vessel diameter and caffeine intake. Women with early onset preeclampsia showed higher glycocalyx degradation, indicated by a larger perfused boundary region (mean: 2.14 [95% CI, 2.05–2.20]), than the remaining groups (mean: normotensive: 1.99 [95% CI, 1.95–2.02], P=0.002; late‐onset preeclampsia: 2.01 [95% CI, 1.96–2.07], P=0.024; gestational diabetes mellitus: 1.97 [95% CI, 1.91–2.04], P=0.004). The percentage of vessels that were filled with red blood cells was significantly lower in early onset preeclampsia. These structural glycocalyx changes were accompanied by elevated plasma concentrations of the glycocalyx components, heparan sulfate proteoglycans and hyaluronic acid, in early onset preeclampsia compared with normotensive pregnancy.ConclusionsGlycocalyx degradation and reduced microvascular perfusion are associated with endothelial dysfunction and activation and vascular injury in early onset preeclampsia.

Glycocalyx Breakdown Is Associated With Severe Disease and Fatal Outcome in Plasmodium falciparum Malaria.

BackgroundInteractions between the endothelium and infected erythrocytes play a major role in the pathogenesis of falciparum malaria, with microvascular dysfunction and parasite sequestration associated with worsening outcomes. The glycocalyx is a carbohydrate-rich layer that lines the endothelium, with multiple roles in vascular homeostasis. The role of the glycocalyx in falciparum malaria and the association with disease severity has not been investigated.MethodsWe prospectively enrolled Indonesian inpatients (aged ≥18 years) with severe (SM) or moderately severe (MSM) falciparum malaria, as defined by World Health Organization criteria, and healthy controls (HCs). On enrollment, blood and urine samples were collected concurrently with measurements of vascular nitric oxide (NO) bioavailability. Urine was assayed for glycocalyx breakdown products (glycosaminoglycans) using a dimethylmethylene blue (GAG-DMMB) and liquid chromatography-tandem mass spectrometry (GAG-MS) assay.ResultsA total of 129 patients (SM = 43, MSM = 57, HC=29) were recruited. GAG-DMMB and GAG-MS (g/mol creatinine) were increased in SM (mean, 95% confidence interval: 3.98, 2.44–5.53 and 6.82, 5.19–8.44) compared to MSM patients (1.78, 1.27–2.29 and 4.87, 4.27–5.46) and HCs (0.22, 0.06–0.37 and 1.24, 0.89–1.59; P < 0.001). In SM patients, GAG-DMMB and GAG-MS were increased in those with a fatal outcome (n = 3; median, interquartile range: 6.72, 3.80–27.87 and 12.15, 7.88–17.20) compared to survivors (n = 39; 3.10, 0.46–4.5 and 4.64, 2.02–15.20; P = 0.03). Glycocalyx degradation was significantly associated with parasite biomass in both MSM (r = 0.48, GAG-DMMB and r = 0.43, GAG-MS; P < 0.001) and SM patients (r = 0.47, P = 0.002 and r = 0.33, P = 0.04) and inversely associated with endothelial NO bioavailability.ConclusionsIncreased endothelial glycocalyx breakdown is associated with severe disease and a fatal outcome in adults with falciparum malaria.

Role of matrix metalloproteinases and histone deacetylase in oxidative stress-induced degradation of the endothelial glycocalyx.

The glycocalyx is crucial for normal endothelial function. It also tethers extracellular superoxide dismutase (SOD3), which protects the endothelium against oxidative damage. Proteolytic enzymes [matrix metalloproteinases (MMPs)] are capable of disrupting endothelial cell surface proteins, such as syndecans, resulting in derangements of the endothelial glycocalyx. We sought to test the role of MMPs in oxidative stress-mediated disruption of the endothelial glycocalyx and examine the effect of pharmacological inhibition of MMPs on mitigating this detrimental effect. We also examined the role of histone deacetylase (HDAC) in the oxidative stress-mediated MMP induction and glycocalyx remodeling. Oxidative stress was experimentally induced in human adipose microvascular endothelial cells using H2O2 and buthionine sulfoximine in the presence and absence of potent MMP and HDAC inhibitors. H2O2 and buthionine sulfoximine resulted in a notable loss of the endothelial glycocalyx; they also increased the expression and proteolytic activity of MMP-2 and MMP-9 and subsequently increased the shedding of syndecan-1 and SOD3 from the endothelial cell surface. MMP upregulation was accompanied by a decline in mRNA and protein levels of their inhibitors, tissue inhibitors of metalloproteinase (TIMPs; TIMP-1 and TIMP-3). Furthermore, oxidative stress induced HDAC activity. Inhibition of MMPs and HDAC reversed syndecan-1 and SOD3 shedding and maintained endothelial glycocalyx integrity. HDAC inhibition increased TIMP expression and reduced MMP expression and activity in endothelial cells. Our findings shed light on MMPs and HDAC as therapeutically targetable mechanisms in oxidative stress-induced glycocalyx remodeling. NEW & NOTEWORTHY Oxidative stress, a hallmark of many diseases, damages the endothelial glycocalyx, resulting in vascular dysfunction. Studying the mechanistic link between oxidative stress and endothelial glycocalyx derangements might help discover new therapeutic targets to preserve vascular function. In this study, we investigated the involvement of matrix metalloproteinases and histone deacetylase in oxidative stress-induced endothelial glycocalyx degradation.

Elevated plasma glypicans are associated with organ failure in patients with infection

BackgroundIncreased vascular permeability is a key feature in the pathophysiology of sepsis and the development of organ failure. Shedding of the endothelial glycocalyx is increasingly being recognized as an important pathophysiological mechanism but at present it is unclear if glypicans contribute to this response. We hypothesized that plasma levels of glypicans (GPC) are elevated in patients with sepsis.MethodsPlasma GPC 1–6 levels were measured by ELISA in 10 patients with sepsis and 10 healthy controls as an initial screening. Plasma GPC 1, 3, and 4 were further measured in a cohort of 184 patients with a clinically confirmed infection. Patients were divided into groups of those who had sepsis and those who had an infection without organ failure. To determine whether plasma glypicans could predict the development of organ failure, patients were further subdivided to those who had organ failure at enrolment and those who developed it after enrollment. The association of plasma GPC 1, 3, and 4 with organ failure and with various markers of inflammation, disease severity, and glycocalyx shedding was investigated.ResultsIn the pilot study, only GPC 1, 3, and 4 were detectable in the plasma of sepsis patients. In the larger cohort, GPC 1, 3, and 4 levels were significantly higher (p < 0.001) in patients with sepsis than in those with infection without organ failure. GPC 1, 3, and 4 were significantly positively correlated with plasma levels of the disease severity markers C-reactive protein, lactate, procalcitonin, and heparin binding protein, and with the marker of glycocalyx degradation syndecan 1. They were significantly negatively correlated with plasma levels of the glycocalyx-protective factors apolipoprotein M and sphingosine-1-phosphate.ConclusionsWe show that GPC 1, 3, and 4 are elevated in plasma of patients with sepsis and correlate with markers of disease severity, systemic inflammation, and glycocalyx damage.

Acute hyperglycemia increases sepsis related glycocalyx degradation and endothelial cellular injury: A microfluidic study

BackgroundHyperglycemia promotes vascular inflammation; however its effect on endothelial dysfunction in sepsis is unknown. Microfluidic devices (MFD) may closely mimic the in vivo endothelial cell microenvironment. We hypothesized that stress glucose concentrations would increase sepsis related endothelial injury/activation. MethodsHuman umbilical vein endothelial cell (HUVEC) monolayers were established in microfluidic channels. TNF was added followed by glucose. Endothelial glycocalyx (EG) integrity was indexed by shedding of the EG components as well as thickness. Endothelial cell (EC) injury/activation was indexed by soluble biomarkers. Intracellular reactive oxygen species (ROS) was by fluorescence. ResultsTNF increased glycocalyx degradation and was associated with biomarkers of EC injury. These vascular barrier derangements were further increased by hyperglycemia. This may be related to increase ROS species generated followed by the combined insults. ConclusionMFD technology may be a useful platform to study endothelial barrier function and stress conditions and allow preclinical assessment of potential therapies.

Sepsis-induced damage to endothelial glycocalyx (literature review)

Glycocalyx is a gel-like layer covering the surface of vascular endothelial cells. It consists of membrane-attached proteoglycans, glycosaminoglycan chains, glycoproteins, and plasma adhesive proteins. Glycocalyx plays a key role in maintaining vascular homeostasis, controls vascular permeability and the tone of the microvasculature, prevents microvascular thrombosis and regulates leukocyte adhesion. In sepsis and septic shock, damage and shedding of glycocalyx occurs. The degradation of glycocalyx is activated by reactive oxygen species and pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1β (IL-1β). The inflammation-mediated degradation of glycocalyx leads to vascular hyperpermeability, unregulated vasodilation, microvascular thrombosis, and enhanced leukocyte adhesion. The inflammation-mediated degradation of glycocalyx leads to vascular hyperpermeability, unregulated vasodilation, microvascular thrombosis, and enhanced leukocyte adhesion. Clinical studies have demonstrated a correlation between the levels of glycocalyx components in the blood and organ dysfunction and mortality in sepsis and septic shock. Inflammation-induced damage to glycocalyx can cause a number of specific clinical effects of sepsis, including acute kidney damage, respiratory failure and liver dysfunction. Infusion therapy is an integral part of the treatment of sepsis, but super-aggressive infusion load methods (leading to hypervolemia) may increase the degradation of glycocalyx. Moreover, some markers of glycocalyx degradation, such as circulating levels of syndecan 1 or heparan sulfate, can be used as markers of endothelial dysfunction and sepsis severity.

Ultrasmall gold nanorods: synthesis and glycocalyx-related permeability in human endothelial cells.

BackgroundClinical data show shed endothelial glycocalyx (GCX) components in blood samples of atherosclerotic patients, linking atherosclerotic development to endothelial GCX integrity. Healthy GCX has pores no >7 nm, and shed GCX has even larger pores. Therefore, we suggest targeting and treating atherosclerosis-prone blood vessels by using nanoscale vehicles to deliver drugs via the nanoscale GCX as it becomes dysfunctional.Materials and methodsTo test our idea, we investigated permeability of nanoparticles in endothelium, as related to a GCX expression. The present work involves nanorods, which are expected to interact with larger portions of endothelial cell (EC) membranes, due to surface area of the nanorod long axis. Conventional nanorod diameters are orders of magnitude larger than the GCX pore size, so we adapted conventional synthesis methods to fabricate ultrasmall gold nanorods (GNRs). Our ultrasmall GNRs have an aspect ratio of 3.4, with a length of 27.9±3.1 nm and a diameter of 8.2±1.4 nm. In addition, we produced GNRs that are biocompatible and fluorescently visible, by coating the surface with functionalized polyethylene glycol and Alexa Fluor 647. To study GNR–GCX interactions, we used human ECs, for species relevance.ResultsUnder life-like flow conditions, the human ECs are densely covered with a 1.3 µm thick layer of GCX, which coincides with minimal GNR permeability. When the GCX is weakened from lack of flow (static culture) or the presence of GCX degradation enzyme in the flow stream, the GCX shows 40% and 60% decreased thickness, respectively. GCX weakness due to lack of flow only slightly increases cellular permeability to GNRs, while GCX weakness due to the presence of enzyme in the flow leads to substantial increase in GNR permeability.ConclusionThese results clarify that the GCX structure is an avenue through which drug-carrying nanoparticles can be delivered for targeting affected blood vessels to treat atherosclerosis.

Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression, localization, and function via glycocalyx degradation

BackgroundEndothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization.MethodsWe studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation.ResultsResults confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177.ConclusionsThis is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.