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Crossing borders: the role of the endothelial glycocalyx and intravascular
haemostasis in vascular complications of diabetes mellitus
Lemkes, B.A.
Publication date
2011
Document Version
Final published version
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Citation for published version (APA):
Lemkes, B. A. (2011). Crossing borders: the role of the endothelial glycocalyx and
intravascular haemostasis in vascular complications of diabetes mellitus.
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Crossing borders:
the role of the endothelial glycocalyx and intravascular haemostasis in vascular complications of diabetes mellitus
Bregtje A. Lemkes
Uitnodiging
voor het bijwonen van de openbare verdediging van
mijn proefschrift
op donderdag 13 oktober 2011 om 12:00u in de Agnietenkapel oudezijds Voorburgwal 231
Amsterdam
U bent van harte uitgenodigd voor de receptie ter plaatse na afloop van de verdediging
Bregtje Lemkes b.a.lemkes@amc.nl
Paranimfen
Lian tjon Soei Len
liantsl@hotmail.com
Emma van der Laan
emmavanderlaan@hotmail.com
Crossing borders:
the role of the endothelial glycocalyx and intravascular haemostasis
in vascular complications of diabetes mellitus
Crossing borders: the role of the endothelial glycocalyx and intravascular haemostasis in vascular complications of diabetes
Academic thesis, University of Amsterdam, Amsterdam, The Netherlands
ISBN: 978-94-6108-217-6
Author: Bregtje A. Lemkes
Lay-out: Nicole Nijhuis, Gildeprint
Print: Gildeprint Drukkerijen, Enschede, The Netherlands
© B.A. Lemkes, Amsterdam 2011
All rights reserved. No part of this publication may be reproduced, stored, or transmitted in any form or by any means, without written permission of the author.
Printing of this thesis was financially supported by: Universiteit van Amsterdam, Stichting Asklepios, sanofi-aventis Netherlands BV.
Crossing borders:
the role of the endothelial glycocalyx and intravascular haemostasis
in vascular complications of diabetes mellitus
ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel op donderdag 13 oktober 2011, te 12:00 uur
door
Bregtje Annelieke Lemkes geboren te Haarlemmermeer
Promotiecommissie:
Promotor: prof. dr. J.B.L. Hoekstra
Co-promotor: dr. F. Holleman Overige leden: prof. dr. J.A. Romijn
prof. dr. E.S.G. Stroes prof. dr. R.O. Schlingemann prof. dr. C.D.A. Stehouwer prof. dr. J.W. Eikelboom Faculteit der Geneeskunde
Contents
Chapter 1 Introduction 7
Chapter 2 Should we judge the endothelium by its cover? 13
On the role of the endothelial glycocalyx in the development of vascular complications in diabetes
Chapter 3 Glucose: a prothrombotic factor? 31
Chapter 4 Mild hyperglycaemia disturbs vascular homeostasis in humans 47
Chapter 5 Effect of sulodexide on endothelial glycocalyx and vascular 67
permeability in patients with type 2 diabetes mellitus
Chapter 6 Improved glycaemic control by insulin therapy ameliorates 87
the prothrombotic state in type 2 diabetes
Chapter 7 Impaired glycocalyx barrier properties and shedding of its 103
constituents in dialysis patients
Chapter 8 The influence of aspirin dose and glycaemic control on platelet 121
inhibition in patients with type 2 diabetes
Chapter 9 The influence of the choice of anticoagulant on the outcome of 137
platelet aggregation tests to monitor aspirin effectiveness
Chapter 10 Summary and final considerations 145
Chapter 11 Nederlandse samenvatting 153
CHAPTER
1
Introduction
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‘Banting’s discovery of insulin only allowed patients with diabetes to live just long enough to develop blindness, renal failure, and coronary disease’
Michael Brownlee, Banting Lecture 20051.
While this statement about one of the most important medical discoveries of the past century may sound unduly harsh, its content unfortunately holds true. In an era with rapid advances in the treatment of hyperglycaemia, including new oral glucose-lowering drugs, biosynthetic insulin analogues and state-of-the-art insulin administration devices, patients with diabetes mellitus are still facing an uncertain fate. Damage to the small blood vessels of the retina will cause severe visual loss in 10% of patients and blindness
in 2%2. Diabetic nephropathy will cause the death of 10-20%.2 Neuropathy will affect
one in two patients with diabetes and, together with impaired blood flow, increases the
risk of foot ulcers and limb amputation2. Ultimately, 50% of patients with diabetes die of
cardiovascular disease2.
When Dr. Brownlee delivered his lecture, over half a century of research into the development of diabetic complications had passed. If anything, the current body of evidence suggests that the pathogenesis of complications in diabetes is multifactorial. This is especially true for the complex entity called type 2 diabetes mellitus, were hyperglycaemia is often accompanied by obesity, insulin resistance, hypertension and dyslipidaemia.
Although each of the diabetic complications has its own tissue-specific pathogenesis, they all share a (micro-or macro)vascular component. Traditionally, hyperglycaemia-induced vascular damage is believed to start with damage to the vascular endothelium, causing endothelial dysfunction. However, the vascular endothelium is lined with a protective border of proteoglycans and adhered glycosaminoglycans, the so-called glycocalyx, and damage to this border is likely to precede endothelial injury. In fact, recent studies by Nieuwdorp and colleagues have shown a direct damaging effect of both
acute hyperglycaemia and type 1 diabetes on the glycocalyx3, 4. These findings have led
to the hypothesis that hyperglycaemia-induced glycocalyx damage is the first step in the development of vascular damage in diabetes and this suggests a new therapeutic target in the prevention of diabetic complications. In chapter 2 of this thesis an overview of the research into this protective layer is presented and its possible role in the development of vascular complications of diabetes is further explored.
While damage to the endothelial glycocalyx represents a new pathogenic pathway in the early stages of vascular disease, diabetes and acute hyperglycaemia are also known to affect the later stages of vascular dysfunction, when an activated coagulation system and aggregating blood platelets cause vascular occlusion. In fact, even though the main
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focus of research has been the development of atherosclerosis, diabetes has been labelled
a ‘prothrombotic condition’, predisposing patients to thrombotic events5, 6. Current
evidence for the effects of diabetes and acute hyperglycaemia on the haemostatic system is reviewed in chapter 3.
As described in the chapters 2 and 3, the negative effects of hyperglycaemia on both the endothelial glycocalyx and the coagulation system have been well established. However, little is known about the exact nature of these effects. At which glucose level do these effects first occur? Are these on-off phenomena occurring at a certain glycaemic threshold, or continuous effects? Could these effects be mediated by oxidative stress, as Brownlee suggested in his lecture? These questions were the focus of the study described in chapter 4.
In chapter 5 the observations regarding damage to the endothelial glycocalyx in patients with type 1 diabetes are expanded to a population with type 2 diabetes and it is explored whether glycocalyx damage might be reversed by oral glycocalyx precursor treatment. This issue is further addressed in chapter 6, where we investigated the reversibility of hyperglycaemia-induced alterations in glycocalyx metabolism, coagulation and fibrinolysis following improved glycaemia through insulin treatment.
Finally, the glycocalyx has also been suggested to be of significance in patients with
end stage renal disease, who are dependent on dialysis7. A suggested loss of glycocalyx
could contribute to the accelerated vascular disease seen in dialysis patients, even in the absence of diabetes, but could also be of importance in peritoneal dialysis by affecting peritoneal transport. However, these hypotheses were all based on experimental in vitro and animal studies and human research was lacking. In chapter 7 we investigated the state of the endothelial glycocalyx in patients on peritoneal or haemodialysis
The prothrombotic state that characterizes patients with type 2 diabetes is the consequence of coagulation activation and fibrinolytic impairment, but is also the result of hyper reactivity of blood platelets. In fact, the propensity of patients with type 2 diabetes towards cardiovascular events has made them likely candidates for primary prevention with an antiplatelet agent. In recent years, several large clinical trials have investigated whether these patients would benefit from low dose aspirin treatment to prevent first cardiovascular events. Surprisingly, these trials have not been able to show
a benefit of aspirin 8-10. Several factors may play a role in the reduced efficacy of aspirin
as an antiplatelet drug in patients with diabetes. Of these, hyperglycaemia is thought to interfere with the mechanism of action of aspirin. Also, patients with diabetes may require higher doses of aspirin due to high baseline platelet reactivity. In chapter 8, the role of glycaemic control as well as aspirin dose on the anti-platelet efficacy of aspirin is examined in a cohort of patients with type 2 diabetes. However, this research into blood platelets is notoriously hampered by suboptimal testing conditions. For one, the
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fect of sulodexide on endothelial glycocalyx and vascular permeability
preservative used in the blood collection tubes may influence the test results through its own anticoagulant properties. Thus, in chapter 9 we describe the effects of various blood preservatives on the platelet function tests used to monitor the effect of aspirin.
This thesis explores two aspects of the development of vascular complications in diabetes that may have been somewhat underexposed before: the role of the endothelial glycocalyx and of haemostasis, each representing a different phase in the pathogenesis of vascular disease. A summary of the most important findings of this thesis and final considerations can be found in chapter 10.
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References
1. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 2005;54(6):1615-1625.
2. World health organisation. Fact sheet Nº 312. 2011.
3. Nieuwdorp M, van Haeften TW, Gouverneur MC et al. Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 2006;55(2):480-486.
4. Nieuwdorp M, Mooij HL, Kroon J et al. Endothelial glycocalyx damage coincides with microalbuminuria in type 1 diabetes. Diabetes 2006;55(4):1127-1132.
5. Carr ME. Diabetes mellitus: a hypercoagulable state. J Diabetes Complications 2001;15(1):44-54. 6. Grant PJ. Diabetes mellitus as a prothrombotic condition. J Intern Med 2007;262(2):157-172. 7. Flessner MF. Endothelial glycocalyx and the peritoneal barrier. Perit Dial Int 2008;28(1):6-12. 8. Belch J, MacCuish A, Campbell I et al. The prevention of progression of arterial disease and diabetes
(POPADAD) trial: factorial randomised placebo controlled trial of aspirin and antioxidants in patients with diabetes and asymptomatic peripheral arterial disease. BMJ 2008;337(oct16_2):a1840. 9. Ogawa H, Nakayama M, Morimoto T et al. Low-dose aspirin for primary prevention of
atherosclerotic events in patients with type 2 diabetes: a randomized controlled trial. JAMA 2008;300(18):2134-2141.
10. Sacco M, Pellegrini F, Roncaglioni MC, Avanzini F, Tognoni G, Nicolucci A. Primary prevention of cardiovascular events with low-dose aspirin and vitamin E in type 2 diabetic patients: results of the Primary Prevention Project (PPP) trial. Diabetes Care 2003;26(12):3264-3272.
CHAPTER
2
Should we judge the endothelium by its cover?
On the role of the endothelial glycocalyx in the
development of vascular complications of diabetes
Bregtje A. Lemkes, Max Nieuwdorp, Joost B.L. Hoekstra and Frits Holleman
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The endothelial glycocalyx in diabetes
15
Patients with diabetes mellitus are characterized by an extraordinary vascular vulnerability. Traditionally, glucose-induced damage to the vascular endothelium is believed to be one of the first steps in the development of vascular damage in this disease. However, in the healthy vessel the endothelium is protected by a layer of highly glycosylated proteins which form a physical barrier between the endothelium and the passing blood flow. Although its presence has been known for half a century, this so-called glycocalyx drew little attention from researchers in the past due to an underestimation of the size. In the last decade it has become clear that its full thickness actually exceeds that of the vascular endothelium. Accumulating research into the functional relevance of the endothelial glycocalyx suggests an important role for the layer in the development of both micro- and macrovascular disease in diabetes mellitus. Here we present an overview of the biochemistry of the intact glycocalyx, and of methods to assess the glycocalyx. We also explore its possible role in the pathophysiology of vascular complications of diabetes.
Historical perspective
Already in 1989 Deckert c.s. hypothesized that a loss of heparan sulphate proteoglycans, one of the main constituents of the glycocalyx, was responsible for the widespread vascular damage
seen in diabetes1. This hypothesis, which was named the “Steno hypothesis”, stated that the
generalized vascular damage occurring in diabetes was largely the result of a genetically determined vulnerability in the composition of the extra cellular matrix. The hypothesis was based on data which showed that the glomerular membrane had a reduced charge selectivity but a preserved size selectivity for macromolecules in early microalbuminuria, a potent predictor of cardiovascular disease in diabetes. It was concluded that this discrepancy was caused by a loss of negative charges on the glomerular basement membrane while the pore size of the membrane was still intact (figure 1). The major determinants of charge on the glomerular membrane are heparan sulphate proteoglycans, which give it a net negative charge. The investigators postulated that the sulphation of heparan sulphates, which takes place enzymatically in the Golgi apparatus, was impaired in patients with diabetes. However, not all patients develop microalbuminuria and it was hypothesized that genetic polymorphisms of these enzymes could cause them to be vulnerable to poor glycaemic control. Since these enzymes are not only present in the endothelial cells of the glomeruli, but also in the mesangium, retina and intima of large vessels, this may explain why microalbuminuria reflects a more widespread vascular damage.
Long before the publication of the Steno paper an interest in the role of glycoproteins in vascular physiology had developed when in the early 1960’s intravital microscopy revealed a plasma free
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was negatively charged, the concept that a thin layer of negatively charged membrane bound proteoglycans and glycoproteins covers the surface of most cells was developed.3 Named after the polysaccharide components of the layer, it was called the “glycocalyx” or “sweet husk”.4 The early findings indicated that the layer was only a few nanometres thick and subsequently little attention was paid to its existence. However, mathematical models comparing the haematocrit measured in capillaries to that measured in large arteries suggested the existence of a much larger endothelial surface layer which was able to interfere with flow in the microvasculature.5 A lack of evidence to support these estimations hampered further investigations into the function and composition of the glycocalyx. However, when in 1996 for the first time the thickness of the glycocalyx could be measured in vivo using a dye-exclusion technique, its thickness proved to be 400 to 500 nm in capillaries6. It then became clear that the glycocalyx must be a dynamic gel-like layer, dependent on the presence of plasma (flow) to maintain its full thickness. This finding established that the endothelial glycocalyx occupied a significant proportion of the microcirculation and research into this component of the vasculature was given a new impulse.
Figure 1. Schematic representation of size selectivity and charge selectivity.
In panel a. both size and charge selectivity are intact. In panel b. charge selectivity is lost due to loss of negative charges on the membrane and the membrane becomes permeable for small negatively charged solutes. In panel c. both charge and size selectivity are lost due to a loss of negative charges on the membrane and an increase in pore size, the membrane becomes permeable.
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The endothelial glycocalyx in diabetes
17
Biochemistry of the intact glycocalyx
The endothelial glycocalyx can be pictured as a highly hydrated layer of a variety of endothelium- and plasma derived macromolecules, which are either bound to the endothelial membrane or to each other (figure 2). Glycoproteins and proteoglycans are
among its main components, serving as anchors to the endothelium7.
Glycoproteins consist of a core protein and covalently linked saccharide side chains of 2
to 15 sugar residues8. This type of glycosylated protein is not exclusive to the glycocalyx;
in fact many extracellular proteins are glycosylated. Some important examples of the glycoproteins present on the endothelial cell surface are the adhesion molecules, such as selectins, integrins and immunoglobulins. All have different functions based on their ability to bind different plasma components and the manner in which they are connected to the endothelial cells.
Figure 2. A schematic overview of the endothelial glycocalyx.
The glycocalyx is visualized as a brush-like layer on the luminal side of the endothelium. The glycoproteins and proteoglycans serve as anchors to the endothelium. Sugar chains of varying length and consistence are linked to the anchor proteins and form the bulk of the layer. Adsorbed plasma proteins are harboured within the layer.
The proteoglycans are a special group of glycoproteins, which are characterized by very
long, unbranched side chains of over 200 sugar residues or glycosaminoglycans (GAG’s)9.
GAG’s contribute greatly to the negative charge of the glycocalyx, due to the occurrence of sulphate uronic acid groups. Three different core proteins with binding sites for GAG’s
can be found on the endothelial cell: the syndecans, glypicans and perlecans10. They are
synthesized in the ribosomes of the endothelial cell, after which the glycosylation takes place in Golgi apparatus. There, a tetrasaccharide is first attached to a serine group on the core protein to serve as a primer for polysaccharide attachment. Then, sugars are added by glycosyltransferases creating long, linear polysaccharides in a varying pattern of disaccharide repeat units, composed of an uronic acid and a hexosamine. After chain
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polymerisation, the growing GAG chain will undergo several modifications including
N-sulphation, O-sulphation and epimerization11. The type of uronic acid or hexosamine
used and the pattern of sulphation determine the class of GAG synthesized12. The structure
and location of negatively charged sulphate groups of the GAG chain will also determine the binding sites for plasma molecules and a small configurational change may alter the binding capabilities of the GAG dramatically. However, when the GAG’s are attached to the endothelial cell surface their function may also be altered by heparanase and sulphatases (SULPH), which can alter both length and sulphation pattern of the GAG’s. The length and geometrical shape of the GAG’s determine the extension of glycocalyx into the vascular lumen and its voluminous aspect. The GAG’s most commonly found in the vasculature are heparan sulphate (50-90%), chondroitin sulphate, dermatan sulphate
and keratan sulphate7. Another GAG abundantly present in the glycocalyx is hyaluronic
acid, also named hyaluronan. Hyaluronan is different from other GAG’s because it is neither sulphated nor covalently linked to a protein during synthesis. The negative charge of the molecule is due to ionisation of the carboxyl groups of the glucuronic acid constituents at physiological pH. Hyaluronan is synthesized by enzymes bound to the cell membrane (hyaluronan synthase 1, 2, 3) and expelled to either the extracellular matrix
or the luminal endothelial side of the membrane13. There it forms a main constituent of
the glycocalyx, contributing greatly to its water binding capabilities. It interacts with other extracellular matrix macromolecules, such as versican and aggrecan; interactions believed to be essential to the structure and assembly of endothelial glycocalyx. Also, hyaluronan interacts with cell surface receptors, notably CD44, and thereby influences
cell behavior14. Degradation of hyaluronan is predominantly done by hyaluronidase-1
and -215.
Assessment of the glycocalyx
As described above, research into this layer is complicated by its collapse when studied
ex vivo, most likely due to dehydration16. Nevertheless, histological staining of the
endothelium had revealed the presence of the glycocalyx. These early studies made
use of dyes such as ruthenium red17or alcian blue, which bind to the negative charges
on anionic glycosylated polymers, or used lectin based stains18. More recent research
has turned to immunostaining with monoclonal antibodies directed against particular
components of the glycocalyx, such as syndecan-1 or heparan sulphates19-21, to more
specifically determine compositional changes in the layer.
However, pathological changes in its dimension or permeability may be missed or underestimated when the layer is not studied under conditions of flow. Therefore, Vink and Duling performed intravital microscopy to visualize hamster cremaster muscle
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The endothelial glycocalyx in diabetes
19
the capillaries and the functional diameter occupied by red blood cells or fluorescently labelled high molecular weight dextrans, which could be diminished by light dye treatment. The authors concluded that between blood and capillary wall a 0.4 to 0.5 µm thick glycocalyx was present. Vink and Duling further explored the permeability of the glycocalyx for different sizes molecules by using fluorescently labelled dextrans of varying molecular size (weight) and charge to determine whether a threshold for
glycocalyx penetration exists23. They found a size dependent half time for the invasion
of the glycocalyx by anionic dextrans, showing that larger molecules took longer to penetrate the glycocalyx. However, a neutral, small molecule such as neutral dextran 40 easily penetrated the glycocalyx.
Since intravital microscopy was unsuitable for use in human research, Nieuwdorp and colleagues made use of these permeability properties of the glycocalyx to expand the research to human studies. They used a tracer dilution technology to determine the difference between the distribution volumes of a glycocalyx permeable tracer (dextran 40) and a glycocalyx impermeable tracer (fluorescently labelled erythrocytes), providing them with a systemic glycocalyx volume. Although this method is laborious and invasive it did result in the first human studies showing the effects of diabetes on systemic
glycocalyx volume24, 25 and the method proved reproducible in healthy volunteers26.
However, its time-consuming nature and underlying complicated physiology, which
may be dependent on more than glycocalyx properties alone27, made it unsuitable
for larger scale studies. At the same time, a method was developed to determine microvascular glycocalyx dimensions by orthogonal polarisation spectroscopy (OPS) or Sidestream Darkfield (SDF) imaging of the sublingual microcirculation. OPS allows the visualization of the smallest sublingual capillaries by visualizing the haemoglobin in
the red blood cells28. Measurement of the diameter of the red blood cell column in these
small vessels provides information on the intravascular space available to erythrocytes. Since leukocytes are much larger than erythrocytes and have been observed to compress
the endothelial glycocalyx transiently while passing through the smallest of capillaries22,
the widening of the erythrocyte column after leukocyte passing became a first measure
for in vivo micro vascular glycocalyx thickness26. Further development of this
non-invasive method to evaluate the microcirculation led to an automized assessment of the dynamic range of the erythrocyte column width. This method is based on the principle that erythrocytes are able to transiently penetrate the endothelial glycocalyx, thereby contributing to the dynamic range of the erythrocyte column. Reduced dynamic range of the erythrocyte column width, determined by the distribution pattern of multiple measurements of erythrocyte column width, can therefore be interpreted a reduction
of glycocalyx dimension29. This method of imaging of the microvasculature can be used
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and currently a trial to assess the peritoneal microvasculature is underway, to determine the relevance of the glycocalyx in peritoneal transport.
To further expand the detection of endothelial glycocalyx properties to other organs, a method was developed to visualize permeation of the endothelial glycocalyx in retinal vessels by double-dye retinal angiography (figure 3). Two dyes conventionally used in ophthalmological practice to visualize retinal and choroidal vessels, fluorescein and indocyanine green, proved to have different intravascular distribution properties. Fluorescein is a small molecule and fills up the entire vascular compartment, while Indocyanine Green rapidly binds to large plasma molecules on administration and is unable to penetrate the endothelial glycocalyx. This difference in intravascular distribution allowed the comparison of the distribution patterns in patients with diabetes and healthy volunteers, revealing a reduced ICG exclusion zone in patients with
diabetes30.
Figure 3. Double dye retinal angiography
A detailed image and schematic overview of fluorescein angiography (left panel-a) and indocyanine green angiography (right panel-b), the yellow square marks a possible measurement site. Indocyanine green does not easily penetrate the glycocalyx because it is bound to plasma proteins, whereas fluorescein does; the difference in diameter could represent glycocalyx thickness.
a. b.
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The endothelial glycocalyx in diabetes
21
Several studies have also used plasma measurement of degradation products of the endothelial glycocalyx to establish shedding from the layer. Increased plasma levels of hyaluronan, hyaluronidase, heparan sulphates and syndecan-1 have all been implicated
as markers of damage to the endothelial glycocalyx19, 21, 24, 25, 31. Further biochemical
studies are essential to determine the biochemical composition of the layer and establish shedding patterns.
In summary, several methods have been tried and tested to gain knowledge about the state of the endothelial glycocalyx in humans. Although direct visualization of the layer in patients remains an elusive goal in the coming years, indirect measures have proven informative on the state of the endothelial glycocalyx in disease.
Effects of diabetes on the glycocalyx
Several pro-atherogenic stimuli affect the endothelial glycocalyx. Smoking, dyslipidaemia, ischemia-reperfusion and inflammatory cytokines have all been described to cause
dimensional changes and shedding of endothelial glycocalyx components19, 21, 31, 32.
Already in 1983 Ceriello described disturbances in the metabolism of GAGs in serum of patients with diabetes, which correlated highly with long lasting hyperglycaemia
(glycosylated haemoglobin A1c)33. In 2005 an animal study in C57BL6 mice showed that
acute hyperglycaemia increased endothelial glycocalyx permeability, most likely by
affecting the hyaluronan component of the glycocalyx34. This was followed by a study
in healthy volunteers which showed that acute hyperglycaemia (15 mmol/l) reduced
total systemic glycocalyx volume by 50% after six hours24. Similar reductions in systemic
glycocalyx volume were found when a state of chronic hyperglycaemia was studied in a group of patients with type 1 diabetes. Systemic glycocalyx volumes were reduced by almost 50%, when compared to healthy volunteers. In patients with type 1 diabetes and
microalbuminuria this loss of glycocalyx volume was even more profound25. Likewise,
patients with type 2 diabetes were found to have significantly reduced sublingual and
retinal glycocalyx dimensions when compared to age matched controls30. In a recent
study, we investigated the glucose level at which these changes occur by performing a stepwise normo-to-hyperglycaemic clamp at glucose levels of 6, 8 and 10 mmol/l in healthy volunteers. We found that, while there was a linear relationship between glucose levels and reactive oxygen species formation even at the lower glucose levels, significant changes to the endothelial glycocalyx only occurred at a glucose level of 10 mmol/l, as reflected by a significant decrease in plasma hyaluronidase activity (unpublished data). The exact mechanism by which these glucose levels lead to structural changes in the glycocalyx is still unknown. Several effects of hyperglycaemia may contribute to damage to the glycocalyx.
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First, the Steno hypothesis suggested that hyperglycaemia influenced the sulphation of heparan sulphates in the Golgi apparatus. The subsequent studies on sulphation patterns in diabetes have mainly focused on the glomerular endothelium, as this was also subject of the Steno hypothesis, and results have been conflicting. While some have reported altered sulphation pattern of heparan sulphates in experimental diabetes others
failed to confirm these findings35-37. It has also been suggested that under hyperglycaemic
conditions GAG size and sulphation pattern remain unchanged, but the number of
heparan sulphate GAG chains present on the core protein is decreased38.
Second, the evidence of the contribution of reactive oxygen species formation to endothelial damage, including damage to the endothelial surface layer, is accumulating. It has been shown that hyperglycaemia induced damage to the endothelial glycocalyx is accompanied by the formation of reactive oxygen species and can also be partially
prevented by addition of potent anti-oxidants24. Hyperglycaemia leads to the formation
of reactive oxygen species by mitochondrial overproduction of superoxide, O2-39. This in
turn activates several intracellular pathways of tissue damage; increased flux through the polyol pathway, intracellular production of advanced glycation end product (AGE) precursors, protein kinase-C (PKC) activation and increased hexosamine pathway
activity39. Although these intracellular processes may affect intracellular proteoglycan
and GAG metabolism in chronic hyperglycaemia, it has also been shown that ROS can directly damage the extracellular matrix. A broad range of GAGs, such as hyaluronan and chondroitin sulphate, can be extensively depolymerised by oxidants in a
dose-dependent manner40, 41, indicating the compositional breakdown of important building
blocks of the glycocalyx. In support of the negative effect of the depolymerisation of hyaluronan it has been shown that fragmentized hyaluronan has pro-inflammatory, immuno-stimulatory and angiogenic effects, while large hyaluronan polymers have
anti-angiogenic, anti-inflammatory and immunosuppressive effects42.
Finally, it has been suggested that hyperglycaemia mainly affects the binding of hyaluronan to the glycocalyx. As described above, the main binding site for hyaluronan is CD 44 and its hyaluronan binding capacity is affected by glycosylation. It is therefore tempting to speculate that glucose levels of 10 mmol/l or higher cause increased glycosylation of CD 44 and reduce its hyaluronan binding capacity.
Although it is clear that hyperglycaemia, acute or chronic, has a damaging effect on the endothelial glycocalyx, less is known about the reversibility of this damage. Although anti-oxidant treatment has been shown to attenuate the effects of hyperglycaemia, the high doses used in these experimental settings are not suitable for clinical use. We recently determined whether improving glucose control by basal insulin therapy in a group of poorly regulated patients with type 2 diabetes would improve hyaluronan
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23
metabolism. Despite marked improvements in both long term glycaemia (HbA1c) and fasting plasma glucose levels, no changes in plasma hyaluronan or hyaluronidase levels were detected (unpublished data). This could mean that either the diabetes-induced damaged to the glycocalyx is not reversible, not solely dependent on hyperglycaemia or that levels of hyperglycaemia were not reduced sufficiently. Moreover, it remains unclear whether measuring plasma levels of circulating components of the glycocalyx is a good reflection of chronic changes to the layer. However, improvements in plasma markers were detected when the effect of offering precursor GlcNAC substrate for GAG synthesis by sulodexide treatment (mammalian derived heparan- and dermatan sulphate) was studied. After 8 weeks of oral treatment, plasma hyaluronidase activity significantly decreased, indicating inhibition of enzymatic glycocalyx degradation by
the increase in substrate bioavailability30. A number of other interventions have shown
beneficial effects in restoration or preservation of the glycocalyx in other disease states,
such as tumour necrosis alpha factor inhibition in acute inflammation31, statin treatment
in familial hypercholesterolemia32 and hydrocortisone43 and anti-thrombin44 in ischemia
reperfusion damage. These interventions help clarify the pathophysiology behind glycocalyx damage in disease, but are not all suitable for routine use in patients at risk, such as the patient with diabetes.
Consequences of glycocalyx damage Increased vascular permeability
As the early studies into the presence of an endothelial glycocalyx have shown, the layer
shows a clear size dependent impermeability23. This quality contributes significantly
to the barrier function of the vascular endothelium45. In vitro studies have shown that
enzymatic degradation of the endothelial glycocalyx increases the permeability of vessels
to solutes of different size.46 This loss of barrier function of the glycocalyx is of functional
relevance in several complications typical for diabetes mellitus.
First, there is strong evidence for a role of the endothelial glycocalyx in maintaining the
glomerular barrier47, 48 and thus preventing urinary albumin loss and renal damage. In
fact, in a study in apolipoprotein E deficient mice endothelial glycocalyx degradation by chronic hyaluronidase infusion led to proteinuria without changes in glomerular
morphology or tubulo-interstitial inflammation49. This indicates a substantial role for the
glycocalyx in upholding the glomerular barrier, even when the filtration barrier formed by the underlying tissue is intact. In diabetes, where urinary albumin loss is considered to be one of the first signals of the development of complications, Nieuwdorp et al have shown that loss of systemic endothelial glycocalyx volume was most profound in
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Next, the loss of barrier properties of the glycocalyx has also been suggested to be relevant for the cardiovascular system. This first became evident when it was shown that removing the glycocalyx from coronary capillary endothelial surface led to myocardial
oedema, which is known to contribute to cardiac dysfunction.50 Moreover, the ‘leakage’ of
large (lipo) proteins may also contribute to the development of atherosclerosis in patients with diabetes. In support, it was shown that a reduction in glycocalyx dimension induced
by an atherogenic diet leads to intimal accumulation of LDL in the carotid artery51 and
transcapillary chylomicron leakage52.
Also, the first studies are emerging that show the damaging effect of diabetes on the
endothelial glycocalyx in the retinal vasculature30, 53 and it is tempting to speculate on the
involvement of the glycocalyx in diabetic retinopathy. Increased vascular permeability is one of the hallmarks of diabetic retinopathy and loss of the barrier function of the glycocalyx may well contribute to this development, which can ultimately lead to serious complications such as macular oedema. In support, an association between increased urinary GAG excretion and the presence of diabetic retinopathy in patients with type 1
diabetes was found54. Nevertheless, studies on the functional properties of the glycocalyx
in the ocular vasculature are still lacking.
Finally, it has been suggested that the barrier properties of the endothelial glycocalyx may influence peritoneal transport during peritoneal dialysis in patients with end stage
renal disease, a major complication of diabetes55.
Impaired signal transduction
The transmembrane domain of the proteoglycans lining the endothelial cells makes them especially suitable for signal transduction from the intravascular lumen to the interior of the endothelial cell. In this respect, there is a specific role for the endothelial glycocalyx in shear stress sensing by the endothelial cell. This is illustrated by an impairment in the release of the vaso-active nitric oxide (NO) in response to shear stress when specific
components of the glycocalyx, such as heparan sulfates56 or hyaluronan57, are degraded
by enzyme treatment. Also, it has been demonstrated that glycocalyx dimension increases
in areas of high flow, most likely via incorporation of hyaluronan into the glycocalyx58.
In contrast, at sites of disturbed vascular flow, such as near arterial bifurcations, the
glycocalyx dimension is significantly less59. These regions are characterized by a high
atherogenic risk and this supports the hypothesis that glycocalyx damage contributes to vascular vulnerability.
This is further supported by a recent study in which hyaluronan synthesis was inhibited over a period of 25 weeks in apolipoprotein E-deficient mice. This caused severe damage to the endothelial glycocalyx and markedly increased aortic plaque burden, disturbed
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The endothelial glycocalyx in diabetes
25
parallel, in patients with diabetes plasma hyaluronan concentrations were found to be a
marker for angiopathy61 and a linear relationship between carotid intima thickness and
increased plasma GAGs was found62.
Inflammation and coagulation activation
The glycocalyx not only functions as a barrier to prevent the transport of solutes and proteins across the endothelium, it also shields receptor molecules such as selectins and integrins from the passing blood and prevents the adhesion of white blood cells to the endothelium. When the glycocalyx is removed or damaged, leukocyte adhesion to the
endothelium quickly follows63-65. In fact, the white blood cell - endothelial cell interaction
in an inflammatory response occurs too swiftly to be part of up regulation of the integrins on the endothelial surface. Rather, it will result from the uncovering of already present
ICAM molecules on the endothelial cell surface.65 This is supported by the finding of
increased leukocyte-endothelium interactions in the ocular vasculature in mice lacking
the heparan sulphate proteoglycan syndecan-166.
Similarly the presence of the glycocalyx prevents the adhesion of activated platelets to the vascular endothelium, as a decrease in glycocalyx dimension has been shown to lead
to platelet adhesion to the endothelium67, 68.
Since increased vascular permeability, a pro-atherogenic and prothrombotic state are all hallmarks of the diabetic vasculature, it seems the endothelial glycocalyx holds special relevance for the development of micro-as well as macrovascular complications in diabetes.
Questions unanswered
Despite recent advances in research into the role of the endothelial glycocalyx in complications of diabetes, several pivotal questions remain to be answered. First, the exact mechanism by which hyperglycaemia damages the endothelial glycocalyx layer and its composition is still speculative. Further studies into oxidative stress induced pathways of glycocalyx damage, altered sulphation patterns of GAGs, epigenetic changes in genes involved in turnover of glycocalyx constituents and changed binding capacity of anchor proteins during hyperglycaemia need to be performed to gain insight in this pathophysiological process. Second, as of yet it is unclear whether endothelial glycocalyx composition and function is uniform throughout all organ systems or whether the glycocalyx has organ specific properties. Our finding of a differentiated endothelial
glycocalyx response to GAG substrate substitution hints towards the latter option30. Also,
as the endothelium can adapt to the demands of the underlying tissue69, it is plausible
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Finally, the contribution of a damaged endothelial glycocalyx to the development of overt vascular disease in diabetes needs to be clarified. Is a loss of glycocalyx merely an indicator of a diseased endothelium, a precursor of endothelial damage, or perhaps both? And is its relevance in protecting the inner lining of the vasculature limited to the early stages of the disease or can it play a role in delaying further damage to an already damaged vascular bed in advanced diabetes?
These questions should be answered to determine which interventional strategies aimed at preserving or even restoring the endothelial glycocalyx would be beneficial to which patients.
In summary, research from the last two decades suggests an important role for the endothelial glycocalyx in upholding vascular integrity. Evidence suggests that the glycocalyx is especially vulnerable in diabetes and preserving this innate protective barrier could become a new strategy in the prevention of vascular complications of this increasingly prevalent disease. The focus of future research is on improving non invasive measurement tools to reliably assess the dimension, permeability and composition of the glycocalyx as well as large prospective trials studying the relationship between cardiovascular disease and endothelial glycocalyx perturbation in humans. Only then the extent of its contribution to vascular disease in patients with diabetes can be truly known and treatment strategies to repair or preserve the layer can be critically evaluated.
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