Glycocalyx Dysfunction Research Articles

Ulinastatin attenuates pulmonary endothelial glycocalyx damage and inhibits endothelial heparanase activity in LPS-induced ARDS

Acute respiratory distress syndrome (ARDS) is a syndrome of acute respiratory failure characterized by major pathologic mechanisms of increased microvascular permeability and inflammation. The glycocalyx lines on the endothelial surface, which determines the vascular permeability, and heparanase play pivotal roles in the degradation of heparan sulfate (HS). HS is the major component of the glycocalyx. The aim of this study is to examine the effects of Ulinastatin (UTI) on vascular permeability and pulmonary endothelial glycocalyx dysfunction induced by lipopolysaccharide (LPS). In our study, C57BL/6 mice and human umbilical vein endothelial cells were stimulated with LPS to induce injury models. After 6 h of LPS stimulation, pulmonary pathological changes, pulmonary edema, and vascular permeability were notably attenuated by UTI. UTI inhibited LPS-induced endothelial glycocalyx destruction and significantly decreased the production of HS as determined by ELISA and immunofluorescence. UTI also reduced the active form of heparanase (50 kDa) expression and heparanase activity. Moreover, lysosome pH was investigated because heparanase (65 kDa) can be reduced easily in its active form at 50 kDa in a low pH environment within lysosome. Results showed that UTI could inhibit LPS-induced pH elevation in lysosome. In conclusion, UTI protects pulmonary endothelial glycocalyx integrity and inhibits heparanase activity during LPS-induced ARDS.

Hyaluronidase 1 Deficiency Preserves Endothelial Function and Glycocalyx Integrity in Early Streptozotocin-Induced Diabetes.

Hyaluronic acid (HA) is a major component of the glycocalyx involved in the vascular wall and endothelial glomerular permeability barrier. Endocytosed hyaluronidase HYAL1 is known to degrade HA into small fragments in different cell types, including endothelial cells. In diabetes, the size and permeability of the glycocalyx are altered. In addition, patients with type 1 diabetes present increased plasma levels of both HA and HYAL1. To investigate the potential implication of HYAL1 in the development of diabetes-induced endothelium dysfunction, we measured endothelial markers, endothelium-dependent vasodilation, arteriolar glycocalyx size, and glomerular barrier properties in wild-type and HYAL1 knockout (KO) mice with or without streptozotocin (STZ)-induced diabetes. We observed that 4 weeks after STZ injections, the lack of HYAL1 1) prevents diabetes-induced increases in soluble P-selectin concentrations and limits the impact of the disease on endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation; 2) increases glycocalyx thickness and maintains glycocalyx structure and HA content during diabetes; and 3) prevents diabetes-induced glomerular barrier dysfunction assessed using the urinary albumin-to-creatinine ratio and urinary ratio of 70- to 40-kDa dextran. Our findings suggest that HYAL1 contributes to endothelial and glycocalyx dysfunction induced by diabetes. HYAL1 inhibitors could be explored as a new therapeutic approach to prevent vascular complications in diabetes.

Toll-like receptors 2 and 4 mediate hyperglycemia induced macrovascular aortic endothelial cell inflammation and perturbation of the endothelial glycocalyx

ObjectivesHyperglycemia-induced inflammation is central to the vascular complications in diabetes. Toll-like receptors (TLRs) are key players in regulating inflammatory responses. There are sparse data on the role of TLR2 and TLR4 in regulating human macrovascular aortic endothelial cells (HMAECs) inflammation and glycocalyx dysfunction under hyperglycemia. We examined the role of TLR2/4 in the above dysfunctions in HMAEC under high glucose (HG) conditions. MethodsHMAECs were treated with high or normal glucose and TLR-2, TLR-4, MyD88, IRF3, TRIF, nuclear NF-κB p65, IL-8, IL-1β, TNF-α, MCP-1, ICAM-1, sVCAM-1, monocyte adhesion to HMAECs, heparan sulfate and hyaluronic acid were measured. ResultsHG upregulated TLR2 and TLR4 mRNA and protein and increased both MyD88 and non-MyD88 pathways, NF-κB p65, inflammatory biomediators, and monocyte adhesion to HMAECs. Heparan sulfate protein expression was reduced and hyaluronic acid secretion was increased on HG exposure. Inhibition of TLR2 and TLR4 signaling by inhibitory peptides and knockdown of TLR-2 and TLR-4 gene expression by siRNA attenuated HG induced inflammation, leukocyte adhesion and glycocalyx dysfunction. An increase in ROS paralleled the increase in TLR-2/4 and antioxidants treatment reduced TLR-2/4 expression and downstream inflammatory biomediators. ConclusionThus hyperglycemia induces HMAEC inflammation and glycocalyx dysfunction through TLR-2/4 pathway activation via increased ROS.

The glycocalyx--linking albuminuria with renal and cardiovascular disease.

Albuminuria is commonly used as a marker of kidney disease progression, but some evidence suggests that albuminuria also contributes to disease progression by inducing renal injury in specific disease conditions. Studies have confirmed that in patients with cardiovascular risk factors, such as diabetes and hypertension, endothelial damage drives progression of kidney disease and cardiovascular disease. A key mechanism that contributes to this process is the loss of the glycocalyx--a polysaccharide gel that lines the luminal endothelial surface and that normally acts as a barrier against albumin filtration. Degradation of the glycocalyx in response to endothelial activation can lead to albuminuria and subsequent renal and vascular inflammation, thus providing a pathophysiological framework for the clinical association of albuminuria with renal and cardiovascular disease progression. In this Review, we examine the likely mechanisms by which glycocalyx dysfunction contributes to kidney injury and explains the link between cardiovascular disease and albuminuria. Evidence suggests that glycocalyx dysfunction is reversible, suggesting that these mechanisms could be considered as therapeutic targets to prevent the progression of renal and cardiovascular disease. This possibility enables the use of existing drugs in new ways, provides an opportunity to develop novel therapies, and indicates that albuminuria should be reconsidered as an end point in clinical trials.

Postoperative fluid management.

Postoperative care units are run by an anesthesiologist or a surgeon, or a team formed of both. Management of postoperative fluid therapy should be done considering both patients' status and intraoperative events. Types of the fluids, amount of the fluid given and timing of the administration are the main topics that determine the fluid management strategy. The main goal of fluid resuscitation is to provide adequate tissue perfusion without harming the patient. The endothelial glycocalyx dysfunction and fluid shift to extracellular compartment should be considered wisely. Fluid management must be done based on patient's body fluid status. Patients who are responsive to fluids can benefit from fluid resuscitation, whereas patients who are not fluid responsive are more likely to suffer complications of over-hydration. Therefore, common use of central venous pressure measurement, which is proved to be inefficient to predict fluid responsiveness, should be avoided. Goal directed strategy is the most rational approach to assess the patient and maintain optimum fluid balance. However, accessible and applicable monitoring tools for determining patient's actual fluid need should be further studied and universalized. The debate around colloids and crystalloids should also be considered with goal directed therapies. Advantages and disadvantages of each solution must be evaluated with the patient's specific condition.

Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor α: a contributor to endothelial cell glycocalyx dysfunction.

The endothelial surface glycocalyx is a hydrated mesh in which proteoglycans are prominent. It is damaged in diseases associated with elevated levels of tumor necrosis factor α (TNF-α). We investigated the mechanism of TNF-α-induced disruption of the glomerular endothelial glycocalyx. We used conditionally immortalized human glomerular endothelial cells (GEnCs), quantitative PCR arrays, Western blotting, immunoprecipitation, immunofluorescence, and dot blots to examine the effects of TNF-α. TNF-α induced syndecan 4 (SDC4) mRNA up-regulation by 2.5-fold, whereas cell surface SDC4 and heparan sulfate (HS) were reduced by 36 and 30%, respectively, and SDC4 and sulfated glycosaminoglycan in the culture medium were increased by 52 and 65%, respectively, indicating TNF-α-induced shedding. Small interfering (siRNA) knockdown of SDC4 (by 52%) caused a corresponding loss of cell surface HS of similar magnitude (38%), and immunoprecipitation demonstrated that SDC4 and HS are shed as intact proteoglycan ectodomains. All of the effects of TNF-α on SDC4 and HS were abrogated by the metalloproteinase (MMP) inhibitor batimastat. Also abrogated was the associated 37% increase in albumin passage across GEnC monolayers. Specific MMP9 knockdown by siRNA similarly blocked TNF-α effects. SDC4 is the predominant HS proteoglycan in the GEnC glycocalyx. TNF-α-induced MMP9-mediated shedding of SDC4 is likely to contribute to the endothelial glycocalyx disruption observed in diabetes and inflammatory states.

Modulation of endothelial glycocalyx structure under inflammatory conditions.

The glycocalyx of the endothelium is an intravascular compartment that creates a barrier between circulating blood and the vessel wall. The glycocalyx is suggested to play an important role in numerous physiological processes including the regulation of vascular permeability, the prevention of the margination of blood cells to the vessel wall, and the transmission of shear stress. Various theoretical models and experimental approaches provide data about changes to the structure and functions of the glycocalyx under various types of inflammatory conditions. These alterations are suggested to promote inflammatory processes in vessels and contribute to the pathogenesis of number of diseases. In this review we summarize current knowledge about the modulation of the glycocalyx under inflammatory conditions and the consequences for the course of inflammation in vessels. The structure and functions of endothelial glycocalyx are briefly discussed in the context of methodological approaches regarding the determination of endothelial glycocalyx and the uncertainty and challenges involved in glycocalyx structure determination. In addition, the modulation of glycocalyx structure under inflammatory conditions and the possible consequences for pathogenesis of selected diseases and medical conditions (in particular, diabetes, atherosclerosis, ischemia/reperfusion, and sepsis) are summarized. Finally, therapeutic strategies to ameliorate glycocalyx dysfunction suggested by various authors are discussed.

The ideal crystalloid – what is ‘balanced’?

This review explores the contemporary definition of the term 'balanced crystalloid' and outlines optimal design features and their underlying rationale. Crystalloid interstitial expansion is unavoidable, but also occurs with colloids when there is endothelial glycocalyx dysfunction. Reduced chloride exposure may lessen kidney dysfunction and injury with a possible mortality benefit. Exact balance from an acid-base perspective is achieved with a crystalloid strong ion difference of 24 mEq/l. This can be done simply by replacing 24 mEq/l of chloride in 0.9% sodium chloride with bicarbonate or organic anion bicarbonate substitutes. Potassium, calcium and magnesium additives are probably unnecessary. Large volumes of mildly hypotonic crystalloids such as lactated Ringer's solution reduce extracellular tonicity in volunteers and increase intracranial pressure in nonbrain-injured experimental animals. A total cation concentration of 154 mmol/l with accompanying anions provides isotonicity. Of the commercial crystalloids, Ringer's acetate solution is close to balanced from both acid-base and tonicity perspectives, and there is little current evidence of acetate toxicity in the context of volume loading, in contrast to renal replacement. The case for balanced crystalloids is growing but unproven. A large randomized controlled trial of balanced crystalloids versus 0.9% sodium chloride is the next step.

The glomerular endothelium emerges as a key player in diabetic nephropathy

The effects of diabetes on glomerular structure and particularly that of the glomerular capillary wall have been extensively documented. By correlation with clinical measurements, Weil and colleagues provide important insights into the functional significance of glomerular structural changes in type 2 diabetes. Podocyte detachment correlates with albumin-to-creatinine ratio, but less strongly than does loss of endothelial fenestrations, which also correlates with reduced glomerular filtration rate. The role of the glomerular endothelium in diabetic nephropathy demands further scrutiny.

P63 Hydrogen sulfide – a novel guardian of the endothelial glycocalyx?

The endothelial glycocalyx occupies substantial space at the luminal surface of the endothelium and serves as protective layer that regulates adhesion of leukocytes to the endothelium. Shedding of the endothelial glycocalyx and increased leukocyte adhesion are common features in inflammatory pathologies pathologies (e.g. ischaemia–reperfusion injury, diabetes) and are mediated in part by ’reactive oxygen species’ (ROS). It has been suggested that H 2 S down-regulates leukocyte adherence via K ATP channels activation on leukocytes and the endothelium as a novel mechanism to limit inflammation. Blood levels of H 2 S are substantially lower in diabetic patients compared to controls suggesting ROS consumption of H 2 S in the diabetic vasculature. However, the role H 2 S in leukocyte adhesion is not understood. We have investigated the effects of GYY4137 in hyperglycaemia (HG) induced endothelial glycocalyx dysfunction and the mechanisms involved in modulation of leukocyte adhesion. Primary bovine retinal endothelial cells (BREC) were exposed to 5.6 mM glucose (NG) and 25 mM glucose (HG) ± GYY4137 (100 μM) for 24 h. Cell based fluorescence assay (CBF) with WGA-FITC was used to study EG changes. Glibenclamide, gliclazide and cromakalim (10 μM) were used to test the role of endothelial K ATP channels in leukostesis. Leukocyte adhesion to BREC was measured in Ibidi perfusion slides (shear stress 1 dyn/cm 2 ). HG-induced endothelial glycocalyx degradation was significantly inhibited by GYY4137 [HG 93 ± 5% vs HG + GYY 107 ± 8%, p < 0.001, n = 12]. Cromakalim, glibenclamide or gliclazide alone had no effect on glycocalyx changes. HG increased leukocyte adhesion was attenuated by GYY4137 [HG 275 ± 1% vs HG GYY 105 ± 11%]. H 2 S driven decrease in leukocyte adhesion was not inhibited by glibenclamide or gliclazide ruling out endothelial K ATP channel activation. H 2 S added simultaneously with HG to BREC pretreated with cromakalim did not affect leukostasis. GYY4137 inhibited HG induced endothelial glycocalyx degradation and leukocyte adhesion highlighting the therapeutic potential for H 2 S donors or the modulation of endogenous H 2 S in preserving endothelial integrity under hyperglycaemic conditions.

Update on the Important New Drug Target in Cardiovascular Medicine – the Vascular Glycocalyx

We reviewed this subject in 2009, pointing out that, to the process of atherothrombosis, glycocalyx dysfunction and damage must be added to the previous known causitive factors. Glycocalyx dysfunction is possibly the very first step in the process of atherothrombosis, being a protective layer between the endothelial cells and the blood. We emphasise the unique feature of glycocalyx mediated vasodilatation in that it is initiated purely by mechanical changes, i.e., changes in vascular wall shear stress, allowing conduit arteries to adjust diameter to demanded blood flow rate. The predeliction of atheroma to sites of low shear stress, the inhibition of the shear response by lumenal hyperglycaemia, and the fact that the response is mediated by nitric oxide (NO), an anti-atheromatous agent has led to the hypothesis that impairment of this pathway is pro-atherogenic. In the microcirculation it has been shown that the glycocalyx must be added to the factors involved in the Starling hypothesis of tissue fluid generation and exchange. As a consequence glycoalyx dysfunction in hyperglycaemia has been postulated to cause oedema and microalbinuria. We suggested that perhaps the arterial glycocalyx will become the most important for future early prevention of people at risk of cardiovascular disease. The advances in this subject since 2009 are the subject of the present review. What has struck us when searching the literature is that research into the glycocalyx has increased very much and now comes from many disciplines; e.g., diabetes, hypertension, bioengineering, physiology, critical care, cardiology, shock. This update is by no means exhaustive, but hopes, again, to bring to the attention of the pharmaceutical industry, the need for grants in the appropriate experimental models.

Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.

Appreciation of the glomerular microcirculation as a specialized microcirculatory bed, rather than as an entirely separate entity, affords important insights into both glomerular and systemic microvascular pathophysiology. In this review we compare regulation of permeability in systemic and glomerular microcirculations, focusing particularly on the role of the endothelial glycocalyx, and consider the implications for disease processes. The luminal surface of vascular endothelium throughout the body is covered with endothelial glycocalyx, comprising surface-anchored proteoglycans, supplemented with adsorbed soluble proteoglycans, glycosaminoglycans and plasma constituents. In both continuous and fenestrated microvessels, this endothelial glycocalyx provides resistance to the transcapillary escape of water and macromolecules, acting as an integral component of the multilayered barrier provided by the walls of these microvessels (ie acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes). Dysfunction of any of these capillary wall components, including the endothelial glycocalyx, can disrupt normal microvascular permeability. Because of its ubiquitous nature, damage to the endothelial glycocalyx alters the permeability of multiple capillary beds: in the glomerulus this is clinically apparent as albuminuria. Generalized damage to the endothelial glycocalyx can therefore manifest as both albuminuria and increased systemic microvascular permeability. This triad of altered endothelial glycocalyx, albuminuria and increased systemic microvascular permeability occurs in a number of important diseases, such as diabetes, with accumulating evidence for a similar phenomenon in ischaemia-reperfusion injury and infectious disease. The detection of albuminuria therefore has implications for the function of the microcirculation as a whole. The importance of the endothelial glycocalyx for other aspects of vascular function/dysfunction, such as mechanotransduction, leukocyte-endothelial interactions and the development of atherosclerosis, indicate that alterations in the endothelial glycocalyx may also be playing a role in the dysfunction of other organs observed in these disease states.

Comparison of sequence of depolarization with terminals distribution of the conducting system in the left ventricle of the pig's heart

We aimed to evaluate the microvascular function in patients with microvascular angina (MVA) by assessing 1) the endothelial glycocalyx barrier properties using sublingual microscopy, and 2) the myocardial perfusion reserve using cardiovascular magnetic resonance (CMR) imaging.Sublingual microscopy was performed in 13 MVA patients (angina pectoris, ST-depression on treadmill testing, normal coronary angiogram) and compared with 2 control groups of 13 volunteers and 14 patients with known obstructive coronary artery disease (CAD). To test the glycocalyx-mediated microvascular responsiveness, the erythrocyte perfused boundary region (PBR) was assessed at baseline and after nitroglycerin challenge.The baseline PBR of MVA patients was similar to controls with CAD (p = 0.72), and larger than in volunteers (p = 0.02). Only the volunteers demonstrated a significant increase in PBR after nitroglycerin (p = 0.03). In the 13 MVA patients, adenosine stress CMR perfusion imaging was performed. Although a significant increase in myocardial perfusion was observed in both the subendocardium and subepicardium during stress, the subendocardial perfusion reserve was significantly lower (p = 0.02). The PBR responsiveness of the sublingual microvasculature showed a strong correlation with the transmural myocardial perfusion reserve (r = 0.86, p < 0.001).Patients with MVA can be characterized by microvascular glycocalyx dysfunction using sublingual microscopy. The strong correlation between sublingual PBR responsiveness and myocardial perfusion reserve suggests that the glycocalyx may play an important role in the regulation of microvascular volume for myocardial perfusion and supports the concept of impaired glycocalyx barrier properties in MVA.

The Important New Drug Target in Cardiovascular Medicine – the Vascular Glycocalyx

The process of atherothrombosis is known to involve endothelial pathology (first drug target), plaque formation (second drug target) and thrombosis (third drug target). However it has recently been postulated that, even before endothelial pathology occurs, the very first step in the process of atherothrombosis is dysfunction of the arterial glycocalyx that lies between the endothelial cells and the blood [1]. So there are really four drug targets, and perhaps the arterial glycocalyx will become the most important for future early prevention of people at risk. We will review the data available on the relationship of glycocalyx dysfunction to risk factors for atherothrombosis and indicate the areas of research that are required to elucidate this important new subject. Up to the present time, hyperglycaemia and oxidised LDL have been identified as causing glycocalyx dysfunction, and we will seek publications on drugs that modify these effects. Attempts are being made to explore the possibility of drug-induced reversal of hyperglycaemia-induced glycocalyx dysfunction. Progress is, however, dependent on grants being made available for work with the essential large animal (pig) experimental model for testing glycocalyx function. Such grants have hitherto not been sufficiently forthcoming, and this needs to be brought urgently to the attention of the pharmaceutical industry.

Hypothesis: arterial glycocalyx dysfunction is the first step in the atherothrombotic process

We present evidence that the 0.5 microm thick gel layer, lining the inner wall of healthy blood vessels, the glycocalyx, is the first line of defence against atherothrombotic disease. All blood vessel linings are coated with this gel, a highly negatively charged structure, rich in anionic sites mostly represented by the sialic acid moieties of glycoproteins and the sulphate and carboxyl groups of heparan-sulphate proteoglycans. Blood flow in arteries is associated with a shear stress at the glycocalyx, which signals the underlying endothelial cells to release nitric oxide (NO), an anti-atherogenic factor. Sites of low shear stress in the arterial tree are more susceptible to atheroma due to lack of NO generation through this mechanism, whereas exercise, by increasing blood flow and shear stress, is protective. We postulate that risk factors for atherothrombosis act by impairing glycocalyx function. That luminal hyperglycaemia causes glycocalyx dysfunction has already been shown; we postulate this to be the first step in the atherothrombotic process in patients with diabetes mellitus and metabolic syndrome (insulin resistance). There is also evidence of glycocalyx defects from exposure to oxidized low-density lipoprotein. We postulate that other risk factors will have a similar action on the glycocalyx as the initiating factor in the disease process, e.g. smoking, hyperlipidaemias and hyperhomocystenaemia. These predictions can now be tested in a large animal model of shear-stress-mediated arterial dilatation.