UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl) UvA-DARE (Digital Academic Repository) Fluid shear stress directly stimulates synthesis of the endothelial glycocalyx : peturbations by hyperglycemia Gouverneur, M.C.L.G. Link to publication Citation for published version (APA): Gouverneur, M. C. L. G. (2006). Fluid shear stress directly stimulates synthesis of the endothelial glycocalyx : peturbations by hyperglycemia. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 31 Dec 2019
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UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Fluid shear stress directly stimulates synthesis of the endothelial glycocalyx : peturbations byhyperglycemia
Gouverneur, M.C.L.G.
Link to publication
Citation for published version (APA):Gouverneur, M. C. L. G. (2006). Fluid shear stress directly stimulates synthesis of the endothelial glycocalyx :peturbations by hyperglycemia.
General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.
disease and stroke are first and second leading cause of death for male and female adults in
developed countries, and are most common viewed as life style diseases. The main CVD risk
factors are raised blood pressure, alcohol consumption, high plasma cholesterol, obesity, and
diabetes.
Diabetes is a chronic disease presented by pathogenic high plasma levels of glucose
(hyperglycemia), which is caused by a deficiency in insulin production (diabetes type 1) or,
more commonly, by an ineffectiveness of insulin’s action to control blood glucose levels
(diabetes type 2). Data shows that approximately 150 million people have diabetes mellitus
worldwide and that this number may well double by the year 2025, which will mostly occur
in developing countries due to population growth, aging, unhealthy diets, obesity and
sedentary lifestyles. The high incidence of cardiovascular disease worldwide and the
upcoming diabetic epidemic, of which 50% will result in mortal cardiovascular
complications, provokes the question what the relationship between these two pathological
schemes is (58).
Despite the prevelance of atherogenic vascular disease and the contribution of diabetic
pathologies, the mechanistic causes of pathogenic vascular damage is still unclear. Currently,
it is generally recognized that the earliest measurable pathogenic functional abnormality of
the vessel wall is endothelial dysfunction (ED), which is characterized by an imbalance
between relaxing and contracting factors, procoagulant and anticoagulant substances, and
between proinflammatory and inflammatory mediators. Although abundant experimental
evidence is now available that the protective properties of the endothelial cell glycocalyx are
Chapter 1 General Introduction
30
essential to maintain optimal endothelial function, studies to unravel the contribution of
glycocalyx perturbation in the development of vascular dysfunction are lacking.
3.2 Focal nature of atherosclerotic lesions and glycocalyx
Atherosclerosis is known to be a local vascular phenomenon that is confined to specific sites
in the vasculature that are exposed to complex and disturbed flow profiles. These sites
demonstrate endothelial dysfunction, as reflected by impaired local production of nitric
oxide, increased leakage of lipids into the vascular wall, recruitment of adhering platelets and
thrombus formation, leukocyte-endothelial adhesion and finally plaque formation.
The glycocalyx has been shown to serve as a mechanosensor of shear stress, mediating shear-
induced release of NO by endothelial cells (55; 56; 59). In fact, selective perturbation of the
glycocalyx leads to increased vascular permeability, attenuated NO availability, and
increased adhesion of leukocytes and platelets. Reconstitution of the glycocalyx results in the
restoration of its barrier and anti adhesive properties (22; 60). In view of the intricate relation
between glycocalyx integrity and vascular homeostasis in experimental models, it has been
postulated that glycocalyx derangement could contribute to increased focal atherogenic
vascular vulnerability in humans (61).
In a recent study, it was hypothesized that endothelial glycocalyx perturbation contributes to
increased vulnerability of the arterial wall exposed to atherogenic risk factors. Glycocalyx
and intima-to-media ratios (IMR) were studied at a low- and a high-risk region within the
murine carotid artery (common region) and internal carotid branch (sinus region) in control
C57BL/6J (C57BL6) and age-matched C57BL/6J/apoE*3-Leiden (apoE*3; on an atherogenic
diet) mice. Electron micrographs revealed significantly thinner glycocalyces [73 vs. 399
nm] and greater IMR [0.096 vs. 0.044] at the sinus region of C57BL6 mice than in the
common region. Thinner glycocalyces [100 vs. 399 nm] and greater IMR [0.071 vs. 0.044]
were also observed in the common region of age-matched apoE*3 mice on an atherogenic
diet for 6 wk vs. C57BL6 mice on a normal diet. Greater IMR were due to greater intima
layers, without significant changes in media layer dimension. In addition, atherogenic diet
resulted in increased endothelial cell thickness at the sinus region [0.85 vs. 0.53 µm] but not
at the common region [0.66 vs. 0.62 µm]. It was therefore concluded that both regional and
Chapter 1 General Introduction
31
diet-induced increases in atherogenic risk are associated with smaller glycocalyx dimensions
and greater IMR and that vascular sites with diminished glycocalyx are more vulnerable to
proinflammatory and atherosclerotic sequelae (62).
3.3 Glycocalyx in diabetes
The cardiovascular complications due to poor hyperglycemic control appear to be a systemic
phenomenon since they are at different sites of the vasculature, ranging from the heart and
eye to the foot (58). Hyperglycemia itself has been shown to induce a wide array of
downstream effects, which adversely affect the protective capacity of the vessel wall (63).
Hyperglycemia has been associated with enhanced endothelial permeability, increased
leukocyte-endothelium adhesion and impaired nitric oxide (NO) bioavailability (64-66),
which are all phenomena that are also associated with changes in glycocalyx composition.
Furthermore, because increased degradation of proteoglycans has previously been
demonstrated in hyperglycemic conditions (67; 68), we felt that the impact of hyperglycemia
on the glycocalyx merits special interest.
These findings let us to the first hypothesis of this thesis that exposure of
vascular endothelium to physiological levels of fluid shear stress is essential
for the synthesis of the endothelial glycocalyx, and that lack of shear stress
induced glycocalyx synthesis contributes to the increased vascular
vulnerability of endothelium at regions exposed to complex flow profiles.
This led us to formulate the second hypothesis of this thesis that
hyperglycemic perturbation of (shear stress induced) glycocalyx synthesis
contributes to increased vascular vulnerability and elevated atherogenic risk
in diabetes.
Chapter 1 General Introduction
32
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36
37
Outline of the Thesis
The hypotheses we want to test in this thesis as stated before are:
(I) The exposure of vascular endothelium to physiological levels of fluid shear stress is
essential for the synthesis of the endothelial glycocalyx, and that the lack of shear stress
induced glycocalyx synthesis contributes to the increased vascular vulnerability of
endothelium at regions exposed to complex flow profiles.
(II) Hyperglycemic perturbation of (shear stress induced) glycocalyx synthesis contributes to
increased vascular vulnerability and elevated atherogenic risk in diabetes.
Chapter 1 gives a general introduction of the endothelial glycocalyx and its role in the
vascular system.
Chapter 2 studies the effect of shear stress on glycocalyx production in cultured endothelial
cells. We show that shear stress increases the incorporation of hyaluronan component of the
endothelial glycocalyx in an in vitro cell culture system.
The effect of hyperglycemia on the hyaluronan production in endothelial cells under flow
conditions described in Chapter 3 shows an attenuated hyaluronan production by
hyperglycemia on endothelial cells exposed to shear stress.
The study in Chapter 4 where we looked at the effect of hyperglycemia on glycocalyx
synthesis shows an initial increase of glycocalyx production followed by a decreased
endothelial glycocalyx content after 6 hours of hyperglycemic exposure, in accordance with
the hyaluronan plasma content profile after 6 hrs of hyperglycemic clamping.
From the clinical study in Chapter 5 we learn that hyperglycemia causes a loss of glycocalyx
volume in healthy human subjects after 6 hours of hyperglycemic clamping.
Chapter 6 gives an overview of the effects of fluid shear stress on the vasculoprotective
properties of the endothelial glycocalyx and discusses the role of the endothelial glycocalyx
in various experimental models, cell culture, animal and human studies and its role in disease
states.
38
Chapter 2
Fluid shear stress stimulates incorporation of hyaluronan
into the endothelial cell glycocalyx
Mirella Gouverneur,1 Jos A. E. Spaan,1 Hans Pannekoek,2
Ruud D. Fontijn2 and Hans Vink1
Departments of 1Medical Physics and 2Medical Biochemistry, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Am J Physiol Heart Circ Physiol 2006 290(1):H458-62
1. Arisaka T, Mitsumata M, Kawasumi M, Tohjima T, Hirose S and Yoshida Y. Effects of shear stress on glycosaminoglycan synthesis in vascular endothelial cells. Ann N Y Acad Sci 748: 543-554, 1995.
2. Constantinescu AA, Vink H and Spaan JA. Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL. Am J Physiol Heart Circ Physiol 280: H1051-H1057, 2001.
3. Constantinescu AA, Vink H and Spaan JA. Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface. Arterioscler Thromb Vasc Biol 23: 1541-1547, 2003.
4. Dekker RJ, van SS, Fontijn RD, Salamanca S, de GP, VanBavel E, Pannekoek H and Horrevoets AJ. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2). Blood 100: 1689-1698, 2002.
5. Elhadj S, Mousa SA and Forsten-Williams K. Chronic pulsatile shear stress impacts synthesis of proteoglycans by endothelial cells: Effect on platelet aggregation and coagulation. J Cell Biochem 86: 239-250, 2002.
6. Esko JD, Stewart TE and Taylor WH. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 82: 3197-3201, 1985.
7. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO and Tarbell JM. Heparan Sulfate Proteoglycan Is a Mechanosensor on Endothelial Cells. Circ Res 2003.
8. Gorog P and Born GV. Uneven distribution of sialic acids on the luminal surface of aortic endothelium. Br J Exp Pathol 64: 418-424, 1983.
9. Haldenby KA, Chappell DC, Winlove CP, Parker KH and Firth JA. Focal and regional variations in the composition of the glycocalyx of large vessel endothelium. J Vasc Res 31: 2-9, 1994.
10. Henry CB and Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. American Journal of Physiology 277: H508-H514, 1999.
11. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA and Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol 285: H722-H726, 2003.
12. Mulivor AW and Lipowsky HH. Role of glycocalyx in leukocyte-endothelial cell adhesion. Am J Physiol Heart Circ Physiol 283: H1282-H1291, 2002.
13. Rosenberg RD. Redesigning heparin. N Engl J Med 344: 673-675, 2001.
14. Sakariassen KS, Aarts PA, de GP, Houdijk WP and Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 102: 522-535, 1983.
16. van den Berg BM, Vink H and Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res 92: 592-594, 2003.
17. Vink H, Constantinescu AA and Spaan JA. Oxidized lipoproteins degrade the endothelial surface layer : implications for platelet-endothelial cell adhesion. Circulation 101: 1500-1502, 2000.
18. Vink H and Duling BR. Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries. Circulation Research 79: 581-589, 1996.
19. Vink H, Duling BR. Capillary endothelial surface layer selectively reduces plasma solute distribution volume. American Journal of Physiology - Heart & Circulatory Physiology 278: H285-H289, 2000.
20. Vink H, Wieringa PA and Spaan JA. Evidence that cell surface charge reduction modifes capillary red cell velocity-flux relationships in hamster cremaster muscle. Journal of Physiology 489: 193-201, 1995.
21. Wang S, Okano M, and Yoshida. Ultrastructure of endothelial cells and lipid deposition on the flow dividers of branchiocephalic and left subclavian arterial bifurcations of the rabbit aorta. J.Jpn.Atheroscler.Soc. 19, 1089-1100. 1991.
22. Weinbaum S, Zhang X, Han Y, Vink H and Cowin SC. Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100: 7988-7995, 2003.
23. Woolf N. The arterial endothelium. In: Pathology of Atherosclerosis, edited by Crawford ST. Butterworths & Co Ltd. London, England., 1982, p. 25-45.
58
Chapter 3
Hyperglycemia attenuates flow induced hyaluronan
production by cultured EC-RF24 endothelial cells
Mirella Gouverneur, Jos A. E. Spaan and Hans Vink
Department of Medical Physics, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
Submitted for publication
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
60
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
61
Abstract The endothelial glycocalyx protects the vascular wall against atherogenic stimuli, and it is suggested that hyperglycemic perturbations of the endothelial glycocalyx contribute to increased vascular vulnerability of diabetic patients. We recently demonstrated that fluid shear stress is an important stimulus of endothelial hyaluronan production, an essential component of the endothelial glycocalyx. In the present study we tested the hypothesis that hyperglycemia impairs shear stress induced endothelial hyaluronan synthesis. Endothelial cells (EC-RF24) were incubated for 18 hours with or without hyperglycemic media (5 or 25mM glucose). Subsequently, the cells were exposed to fluid shear stresses of 1, 5 and 10 dynes/cm2 under normo- or hyperglycemic conditions. Samples were taken from the media solutions at t = 2 hours to determine the initial amount of hyaluronan released from the endothelium upon initiation of fluid shear stress. Measurements of hyaluronan levels at later time points up to 9 hours during exposure to fluid shear stress were performed to determine the amount of shear stress induced endothelial hyaluronan release. Hyaluronan levels were determined using a commercial hyaluronan ELISA kit. Initiation of fluid shear stress resulted in a rapid, initial endothelial release of hyaluronan, independent of the level of fluid shear stress. Initial release of hyaluronan from normoglycemic cells averaged 2.7 ± 0.9 ng / 10000 cells (pooled data). Exposure of endothelial cells to hyperglycemic conditions for 18 hours increased the initial shear stress induced hyaluronan release to 6.3 ± 1.5 ng / 10000 cells (P < 0.05). Continued exposure of normoglycemic endothelial cells to fluid shear stress of 10 dynes/cm2 induced endothelial hyaluronan release at a rate of 18.3 ± 1.4 ng / 10000 cells/6h, which is significantly greater than hyaluronan release rates of 5.5 ± 5.1 and 0.2 ± 2.3 ng / 10000 cells/6h at fluid shear stress levels of 5 and 1 dynes/cm2, respectively. Hyperglycemia significantly attenuated endothelial hyaluronan release rate at 10 dynes/cm2 to 7.6 ± 6.7 ng / 10000 cells/6h. Exposure of cultured endothelial cells to hyperglycemic conditions for 18 hours doubles the initial shedding of hyaluronan from the endothelial surface upon the start of fluid shear stress exposure. Hyperglycemic hyaluronan shedding is accompanied by impaired shear stress induced endothelial hyaluronan synthesis at 10 dynes/cm2, which is consistent with the hypothesis that hyperglycemic loss of hyaluronan from the endothelial glycocalyx impairs flow dependent endothelial hyaluronan synthesis by impaired mechano-transduction of fluid shear stress.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
62
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
63
3.1 Introduction
Patients with diabetes mellitus are characterized by increased vascular vulnerability, leading
to accelerated macrovascular disease such as atherogenesis as well as microvascular
complications (10). Hyperglycemia and poor hyperglycemic control are considered as
important contributors to higher cardiovascular complication incidence in the diabetic state
(1). The in vivo endothelial glycocalyx is a highly negatively charged protective barrier
between endothelial cells and flowing blood consisting of glycoproteins, proteoglycans and
associated glycosaminoglycans and plasma proteins (27). Hyaluronan glycosaminoglycans,
one of the major constituents of the glycocalyx, are of crucial importance for maintaining
endothelial barrier properties and mechanotransduction of fluid shear stress (11).
Furthermore, it has recently been shown that fluid shear stress stimulates incorporation of
hyaluronan into the endothelial cell glycocalyx (8). The dimension of the endothelial
glycocalyx exceeds those of endothelial adhesive molecules (25), which may explain its
potent anti-adhesive effects towards both leukocytes and platelets (3; 4; 18; 20; 21).
Perturbations of the endothelial glycocalyx, such as hyperglycemia (26) are coming more into
focus as playing a major role in the initiation of vascular complications (15). The hypothesis
of this study therefore is whether hyperglycemia impairs shear stress induced hyaluronan
synthesis.
3.2 Materials and Methods
Chemicals
M199 media, L-glutamine, antibiotic-antimycotic and trypsin were obtained from Gibco-
BRL, PBS pH: 7.4 from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker.
The following chemicals were obtained from Sigma; heparin, endothelial cell growth
supplement (ECGS). D(+)glucose was obtained from Merck. The Hyaluronan Enzyme-linked
Immunosorbent Assay kit was obtained form Echelon biosciences incorporated, Salt Lake
City, USA. Fibronectin was a kind gift from Central Laboratory for Blood transfusion (CLB),
Amsterdam, The Netherlands.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
64
Cell culture
EC-RF24 cell line, human umbilical vein endothelial cells immortalized with an
amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7
DNA (6). The cells were grown on 10µg/ml fibronectin-coated cell culture flasks in M199
media supplemented with 20% heat-inactivated fetal bovine serum, 50µg/ml heparin and
12.5µg/ml endothelial cell growth supplement, 0.2mmol/l L-glutamine and 100U/ml
Penicillin-G, 100U/ml Streptomycin sulfate, 25µµg/ml Amphotericin-B at 37oC in 5% CO2.
Parallel flow chamber
The parallel flow perfusion chamber system used is described in detail by Sakariassen KS et
al (19). The parallel flow perfusion chamber was generously provided by Prof. P.G. de Groot,
Dept. Hematology, University Medical Center, The Netherlands(9).
Short-term shear stress exposure under normo (5mM) and hyperglycemic (25mM) conditions
The cells were seeded on fibronectin coated thermanox cover slips and attached to
confluency for 18 hours in complete M199 media (approximately 2x105cells/thermanox)
under normo (5mM) and hyperglycemic (25mM) conditions, subsequently, the cells were
placed in parallel flow chamber and exposed to steady laminar flow at shear stresses of 1, 5
and 10dynes/cm2. At times 0, 2, 4, 6 and 9 hours an aliquot of 250µl (of total 20ml) was
taken from the media and stored at 4oC.
Hyaluronan content
Hyaluronan mass was determined using enzyme linked immunosorbent assay kit,
commercially available from Echelon biosciences incorporated. The principle is based on
competitive ELISA assay in which the colorimetric signal is inversely proportional to the
amount of hyaluronan present in the sample.
Statistical analysis
For statistical analysis, two-way unpaired t-tests were used. A value of P < 0.05 was
considered statistically significant. Values are means ± SE.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
65
3.3 Results
Effect of hyperglycemia on initial hyaluronan shedding from endothelial cells
EC-RF24 cells were seeded on fibronectin-coated cover slips and grown to confluency (105
cells/cover slip). The cells were exposed to normoglycemic (5mM glucose) or hyperglycemic
(25mM glucose) media for 18 hours. Upon initiation of endothelial fluid shear stress
exposure, hyaluronan content in media collected at t = 2 hours from hyperglycemic cells was
significantly elevated compared to the level of initial hyaluronan shedding from
normoglycemic endothelial cells, being 6.3 ± 1.5 and 2.7 ± 0.9 ng / 10000 cells (P < 0.05),
respectively (figure 3.1).
Effect of shear stress levels on endothelial hyaluronan synthesis under normo- and
hyperglycemic conditions
Following the initial hyaluronan shedding, exposure of normo- and hyperglycemic
endothelial cells to fluid shear stresses of 1, 5 and 10dynes/cm2 was continued up to 7 more
hours under normoglycemic (5mM) and hyperglycemic (25mM) media conditions. At low
Figure 3.1: Initial hyaluronan shedding under shear conditions: Effect of hyperglycemia. The initial
hyaluronan shedding is significantly higher under the hyperglycemic condition compared to the
normoglycemic condition at all shear levels (6.3±1.5 versus 2.7±0.9 ng hyaluronan/10000cells) (P<0.05).
Initial endothelial hyaluronan shedding from flow start till t = 2h
0
5
10
15
Initi
al e
ndot
helia
l HA
she
ddin
g (n
g/ 1
0000
cel
ls)
Normoglycemia
Hyperglycemia
Initial endothelial hyaluronan shedding from flow start till t = 2h
0
5
10
15
Initi
al e
ndot
helia
l HA
she
ddin
g (n
g/ 1
0000
cel
ls)
Normoglycemia
Hyperglycemia
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
66
shear stress levels of 1 and 5 dynes/cm2, both normo- and hyperglycemic conditions do not
significantly stimulate endothelial hyaluronan shedding into the media over time (Figure 3.2
and 3.4).
At a shear level of 10dynes/cm2, normoglycemic endothelial cells are stimulated to release
hyaluronan into the media at a rate of 18.3 ± 1.4 ng / 10000 cells/6h, which is significantly
greater than hyaluronan synthesis at 10 dynes/cm2 by hyperglycemic endothelial cells, being
7.6 ± 6.7 ng / 10000 cells/6h (Figure 3.3 and 3.4).
Figure 3.2: Hyaluronan release under 1 dyne/cm2 shear stress: Effect of hyperglycemia. Endothelial
cell exposed to a low shear stress of 1 dyne/cm2 is not significantly changed after 6 hours and
unaltered by hyperglycemic condition.
Effect of hyperglycemia on 1 dynes/cm2 shear stress induced endothelial hyaluronan release
y = -0.4x + 7.2
y = 0.1x + 3.2
0
5
10
15
0 2 4 6 8
Duration of 1 dynes/cm2 shear stress exposure (hours)
Endo
thel
ial h
yalu
rona
n(n
g/ 1
0000
cel
ls)
NormoGlycemia HyperGlycemia
Initial hyaluronan shedding upon flow start
Effect of hyperglycemia on 1 dynes/cm2 shear stress induced endothelial hyaluronan release
y = -0.4x + 7.2
y = 0.1x + 3.2
0
5
10
15
0 2 4 6 8
Duration of 1 dynes/cm2 shear stress exposure (hours)
Endo
thel
ial h
yalu
rona
n(n
g/ 1
0000
cel
ls)
NormoGlycemia HyperGlycemia
Initial hyaluronan shedding upon flow start
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
67
Figure 3.3: Hyaluronan release under 10 dynes/cm2 shear stress: Effect of hyperglycemia. Endothelial cell
exposed to a shear stress of 10 dyne/cm2 is significantly increased over time (21.4±1.4 ng
hyaluronan/10000cells) (P<0.05) and attenuated by the hyperglycemic condition (8.9±5.3) (P<0.05).
Figure 3.4: Hyaluronan shedding under hyperglycemia: Effect of shear stress level. At shear stress levels of 1
and 5 dynes/cm2, the hyaluronan shedding is not significantly increased over time and the hyperglycemic
condition shows no effect on hyaluronan shedding. At shear level of 10 dynes/cm2, the hyaluronan shedding is
significantly increased over time and under hyperglycemic condition the hyaluronan shedding is depressed (18.3
± 1.4 versus 7.6 ± 6.7 ng / 10000 cells) (P<0.05).
Effect of hyperglycemia on 10 dynes/cm2 shear stress induced endothelial hyaluronan release
y = 3.1x - 0.4
y = 1.3x - 0.5
0
5
10
15
20
25
0 2 4 6 8Time - T2 (hours)
Endo
thel
ial h
yalu
rona
n -i
nitia
l HA
(n
g/ 1
0000
cel
ls)
NormoglycemiaHyperglycemia
Effect of hyperglycemia on 10 dynes/cm2 shear stress induced endothelial hyaluronan release
y = 3.1x - 0.4
y = 1.3x - 0.5
0
5
10
15
20
25
0 2 4 6 8Time - T2 (hours)
Endo
thel
ial h
yalu
rona
n -i
nitia
l HA
(n
g/ 1
0000
cel
ls)
NormoglycemiaHyperglycemia
Shear stress induced hyaluronan release from normo-and hyperglycemic endothelial cells
-5
0
5
10
15
20
25
1 dynes/cm2 5 dynes/cm2 10 dynes/cm2
Endo
thel
ial h
yalu
rona
n re
leas
e (n
g/ 1
0000
cel
ls /
6hou
rs)
NormoglycemiaHyperglycemia
*
Shear stress induced hyaluronan release from normo-and hyperglycemic endothelial cells
-5
0
5
10
15
20
25
1 dynes/cm2 5 dynes/cm2 10 dynes/cm2
Endo
thel
ial h
yalu
rona
n re
leas
e (n
g/ 1
0000
cel
ls /
6hou
rs)
NormoglycemiaHyperglycemia
*
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
68
3.4 Discussion
The most important finding of the current study is that hyperglycemia impairs flow mediated
endothelial cell hyaluronan synthesis at physiological shear stress of 10dynes/cm2. In
addition, it is demonstrated that low shear stress levels of 1 dyne/cm2 are not sufficient to
stimulate endothelial hyaluronan synthesis, which is consistent with previous findings of very
little endothelial cell hyaluronan synthesis under static conditions (8). Furthermore, we report
that overnight incubation of static endothelial cells with hyperglycemic medium is
accompanied by greater amounts of hyaluronan that are initially released from the endothelial
surface upon initiation of fluid shear stress exposure.
Acute effect of hyperglycemia on hyaluronan synthesis
Our findings are consistent with reports by others using cell, animal and human models on
the effects of acute hyperglycemia on endothelial cell hyaluronan in relation to vascular
complications. Studies show that hyaluronan is shedded by oxygen radicals induced by
hyperglycemia in cultured endothelial cells (7), and the effect of advanced glycation end
products (AGEs) on vitreous gel of the eye showed that in addition to light exposure, AGEs
promote the decrease of MW hyaluronan in vitreous of diabetic patients (13). Type 2 diabetic
rats show 2-fold increase in hyaluronidase (12) and increased shedding of hyaluronan in
plasma compared to healthy controls (2). Furthermore, short-term hyperglycemia increases
endothelial glycocalyx permeability with acute capillary shutdown (30). Additionally, Wang
and Hascall show that hyaluronic structures synthesized by rat mesengial cells in response to
hyperglycemia induce monocyte adhesion (28). Recently, human studies show that
hyperglycemic clamping elicits a profound reduction in glycocalyx volume coinciding with
increased circulating plasma levels of glycocalyx constituents like hyaluronan, which is
consistent with release of glycocalyx constituents into the circulation (15).
Mechano-transduction of shear stress and the glycocalyx.
Presently, the mechanism by which endothelial cells sense shear stress is incompletely
understood. In vivo experiments have shown a decreased glycocalyx in of the vasculature
exposed to disturbed shear stress regions, which are considered at high- atherogenic risk (24).
In addition, static endothelial cell shown decreased hyaluronan staining compared to flow
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
synthesis, but recent studies have failed to demonstrate significant differences in HAS gene
activation at undisturbed versus disturbed shear stress regions (16). Alternatively, the
hyaluronan synthase enzyme HAS is located at the plasma membrane of endothelial cells. It
is therefore possible that mechanotransduction of fluid shear stress can directly stimulate
HAS activity.
We and others have demonstrated that an intact glycocalyx is required for endothelial cell
release of nitric oxide (NO) in response to shear stress. Studies using glycocalyx component
removing enzymes, hyaluronidase (14) and heparitinase (5) neuraminidase (17) to disturb the
endothelial glycocalyx, show these components to be participating in mechanosensing that
mediates NO production in response to shear stress. In addition, compromising the
endothelial glycocalyx or endothelial surface layer by enzyme treatment and/or decreasing
environmental protein concentration show a decreased cytoskeletal reorganization of the
endothelial cells in response to shear stress (23), elaborating on the findings that the core
proteins in the bush-like glycocalyx structures have a flexural rigidity that is sufficiently stiff
to serve as a molecular filter for plasma proteins and shows to be an exquisitely designed
transducer of fluid shearing stresses (22; 29). Therefore, our finding of hyperglycemic
induced hyaluronan shedding prior to the shear stress exposure is consistent with the
possibility with impaired mechanotransduction of shear stress due to hyperglycemic
glycocalyx perturbations. In support, in a recent study we demonstrated that hyperglycemic
shedding of glycocalyx hyaluronan in humans is associated with impaired flow mediated
arterial dilatation (15).
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
70
References
1. Ceriello A. Impaired glucose tolerance and cardiovascular disease: The possible role of post-prandial hyperglycemia. American Heart Journal 147: 803-807, 2004.
2. Chajara A, Raoudi M, Delpech B, Leroy M, Basuyau JP and Levesque H. Circulating hyaluronan and hyaluronidase are increased in diabetic rats. Diabetologia 43: 387-388, 2000.
3. Constantinescu AA, Vink H and Spaan JA. Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface. Arterioscler Thromb Vasc Biol 23: 1541-1547, 2003.
4. Fibbi G, Vannucchi S, cavallini P, Del Rosso M, Pasquali F, Cappelletti R and Chiarugi V. Involvement of chondroitin sulfate in preventing adhesive cellular interactions. Biochim Biophys Acta 762: 512-518, 1983.
5. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO and Tarbell JM. Heparan Sulfate Proteoglycan Is a Mechanosensor on Endothelial Cells. Circ Res 2003.
6. Fontijn R, Hop C, Brinkman HJ, Slater R, Westerveld A, van MJ and Pannekoek H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp Cell Res 216: 199-207, 1995.
7. Giardino I, Edelstein D and Brownlee M. BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells. J Clin Invest 97: 1422-1428, 1996.
8. Gouverneur M, Spaan JAE, Pannekoek H, Fontijn RD and Vink H. Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx. American Journal of Physiology-Heart and Circulatory Physiology 290: H458-H462, 2006.
9. Grimm J, Keller R and de Groot PG. Laminar flow induces cell polarity and leads to rearrangement of proteoglycan metabolism in endothelial cells. Thromb Haemost 60: 437-441, 1988.
10. Haffner SM, Lehto S, Ronnemaa T, Pyorala K and Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. New England Journal of Medicine 339: 229-234, 1998.
11. Henry CB and Duling BR. TNF-alpha increases entry of macromolecules into luminal endothelial cell glycocalyx. American Journal of Physiology - Heart & Circulatory Physiology 279: H2815-H2823, 2000.
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12. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N and Takahashi T. Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats. Glycobiology 14: 65-72, 2004.
13. Katsumura C, Sugiyama T, Nakamura K, Obayashi H, Hasegawa G and Ikeda T. Effects of advanced glycation endo products on hyaluronan proteolysis: A new mechanism of diabetic vitreopathy. Ophthalmic research 36: 327-331, 2004.
14. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA and Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol 285: H722-H726, 2003.
15. Nieuwdorp M, van Haeften TM, Gouverneur MCLG, Mooij HL, van Lieshout MHP, Levi M, Meijers JCM, Holleman F, Hoekstra JBL, Vink H, Kastelein JJP and Stroes ESG. Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 55: 480-486, 2006.
16. Passerini AG, Polacek DC, Shi C, Francesco NM, Manduchi E, Grant GR, Pritchard WF, Powell S, Chang GY, Stoeckert CJJ and Davies PF. Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta. Proc Natl Acad Sci U S A 101: 2482-2487, 2004.
17. Pohl U, Herlan K, Huang A and Bassenge E. Edrf-Mediated Shear-Induced Dilation Opposes Myogenic Vasoconstriction in Small Rabbit Arteries. American Journal of Physiology 261: H2016-H2023, 1991.
18. Sabri S, Soler M, Foa C, Pierres A, Benoliel AM and Bongrand P. Glycocalyx modulation is a physiological means of regulating cell adhesion. Journal of Cell Science 113: 1589-1600, 2000.
19. Sakariassen KS, Aarts PA, de GP, Houdijk WP and Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 102: 522-535, 1983.
20. Silvestro L, Ruikun C, Sommer F, Duc TM, Biancone L, Montrucchio G and Camussi G. Platelet-Activating Factor-Induced Endothelial-Cell Expression of Adhesion Molecules and Modulation of Surface Glycocalyx, Evaluated by Electron-Spectroscopy Chemical-Analysis. Seminars in Thrombosis and Hemostasis 20: 214-222, 1994.
21. Soler M, Desplat-Jego S, Vacher B, Ponsonnet L, Fraterno M, Bongrand P, Martin JM and Foa C. Adhesion-related glycocalyx study: quantitative approach with imaging-spectrum in the energy filtering transmission electron microscope (EFTEM). FEBS Letters 429: 89-94, 1998.
22. Squire JM, Chew M, Nneji G, Neal C, Barry J and Michel C. Quasi-periodic substructure in the microvessel endothelial glycocalyx: a possible explanation for molecular filtering? J Struct Biol 136: 239-255, 2001.
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23. Thi MM, Tarbell JM, Weinbaum S and Spray DC. The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A "bumper-car" model. Proceedings of the National Academy of Sciences of the United States of America 101: 16483-16488, 2004.
24. van den Berg BM, Spaan JAE, Rolf TM and Vink H. Atherogenic region and diet diminish glycocalyx dimension and increase intima-to-media ratios at murine carotid artery bifurcation. American Journal of Physiology-Heart and Circulatory Physiology 290: H915-H920, 2006.
25. van Haaren PMA, VanBavel E, Vink H and Spaan JAE. Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy. American Journal of Physiology-Heart and Circulatory Physiology 285: H2848-H2856, 2003.
26. Vink H, Constantinescu AA and Spaan JA. Oxidized lipoproteins degrade the endothelial surface layer : implications for platelet-endothelial cell adhesion. Circulation 101: 1500-1502, 2000.
27. Vink H, Wieringa PA and Spaan JA. Evidence that cell surface charge reduction modifes capillary red cell velocity-flux relationships in hamster cremaster muscle. Journal of Physiology 489: 193-201, 1995.
28. Wang A and Hascall VC. Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion. J Biol Chem 279: 10279-10285, 2004.
29. Weinbaum S, Zhang X, Han Y, Vink H and Cowin SC. Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100: 7988-7995, 2003.
30. Zuurbier CJ, Demirci C, Koeman A, Vink H and Ince C. Short-term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases lineal density of capillaries with flowing red blood cells. Journal of Applied Physiology 99: 1471-1476, 2005.
Chapter 4
Hyperglycemic glycocalyx loss is secondary to an initial
transient increase in glycocalyx synthesis
Mirella Gouverneur1, Max Nieuwdorp2, Jos AE Spaan1,
Erik Stroes2 and Hans Vink1
Departments of Medical Physics1 and Vascular Medicine2, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Submitted for publication
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
74
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
75
Abstract
Recently, we demonstrated that exposure of healthy volunteers to 6 hours of
normoinsulinemic hyperglycemia results in loss of systemic glycocalyx volume. In the
present study, we determined the effect of hyperglycemia on cultured endothelial cell
glycocalyx synthesis and shedding. In the present study we determined the effect of acute
hyperglycemia (25mM glucose for 6 hours) on the biochemical incorporation of 3H-
glucosamine and 35S-sulfate into the glycocalyx of venular endothelial cells. In addition, the
dynamics of hyperglycemic glycocalyx shedding is compared with the dynamics of plasma
levels of glycocalyx hyaluronan in healthy volunteers during 6 hours of hyperglycemic
clamping. Hyperglycemia stimulated shedding of glucosamine containing
glycosaminoglycans from the endothelial by 40% compared to normoglycemic endothelial
cells (P<0.05). Hyperglycemic shedding was accompanied by a rapid, transient 50% increase
in glycocalyx synthesis within the first hour of hyperglycemia (P<0.05). After 1 hour,
glycocalyx synthesis normalized to normoglycemic levels. In contrast, hyperglycemic
glycocalyx shedding maintained increased during the 6 hour exposure to hyperglycemia in a
similar fashion as recently determined plasma levels of glycocalyx hyaluronan in human
volunteers exposed to acute hyperglycemia. The elevated level of glycocalyx shedding
relative to normalized glycocalyx synthesis, resulted in a net reduction of glycocalyx
glucosamine content by 20% compared to normoglycemic controls (P<0.05). Hyperglycemia
induces a transient increase in glycocalyx synthesis, which is followed by a maintained
elevated level of glycocalyx shedding, resulted in net loss of glycocalyx from the surface of
endothelial cells after 6 hours of hyperglycemia.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
76
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
77
4.1 Introduction
The main contributor to higher cardiovascular complication incidence in the diabetic state is
increased hyperglycemia and poor hyperglycemic control (1). Evidence has shown that
glucose serum levels 2 hours after oral challenge (“hyperglycemic spikes”) is a powerful
mediator of cardiovascular risk (4) Infarction meta analysis show a continuous correlation
between glucose serum levels and cardiovascular complications even in subjects without
diabetes (3). Furthermore, hyperglycemia has been associated with increased vascular
permeability and increased platelet and leukocyte adhesion (4; 12). The in vivo endothelial
glycocalyx is a highly negatively charged protective barrier between endothelial cells and
flowing blood consisting of glycoproteins, proteoglycans and associated glycosaminoglycans
and plasma proteins (21). Its functions include establishing permeability barrier (20), binding
anticoagulation factors (15), modulating leukocyte interactions with the endothelium (6; 13),
limiting myocardial edema (19) and it plays a role in mechano shear sensing (23). Recently
we demonstrated that 6 hours of hyperglycemia decreases systemic glycocalyx volume in
healthy volunteers (14). In this study we tested the effect of acute hyperglycemia on
glycocalyx of cultured endothelial cells and compared the dynamics of hyperglycemic
glycocalyx shedding with dynamics of plasma hyaluronan concentration in hyperglycemic
humans.
4.2 Materials and Methods
Chemicals
M199 media, L-glutamine, antibiotic-antimycotic and trypsin were obtained from Gibco-
BRL, PBS pH: 7.4 from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker.
The following chemicals were obtained from Sigma; heparin, endothelial cell growth
supplement (ECGS). D(+)glucose was obtained from Merck. The radiochemicals 6-
[3H]glucosamine (specific activity: ~25-40Ci/mmol) and carrier-free Na[35S]O4 (specific
activity: ~43Ci/mgS) were purchased from ICN. Fibronectin was a kind gift from Central
Laboratory for Blood transfusion (CLB), Amsterdam, The Netherlands.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
78
Cell culture
EC-RF24 cell line, human umbilical vein endothelial cells immortalized with an
amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7
DNA (8). The cells were grown on 10µg/ml fibronectin-coated cell culture flasks in M199
media supplemented with 20% heat-inactivated fetal bovine serum, 50µg/ml heparin and
12.5µg/ml endothelial cell growth supplement, 0.2mmol/l L-glutamine and 100U/ml
Penicillin-G, 100U/ml Streptomycin sulfate, 25µg/ml Amphotericin-B at 37oC in 5% CO2.
Radioactive incorporation studies and hyperglycemic stimulus
The cells were seeded on fibronectin coated thermanox cover slips and attached to
confluency for 2 hours in M199 media without antibiotics (approximately
2x105cells/thermanox). The cells were incubated for 24 hours in complete media M199
without antibiotics, supplemented with 40µCi/ml 6-[3H] glucosamine and 50µCi/ml
Na2[35S]O4 (7). After 24 hours the cells were exposed to normoglycemic (5mM) and
hyperglycemic (25mM) media for 6 hour, during which samples were taken at time points 5,
27, 80, 180, 360min. After 6 hours, the cells were treated with 0.05% trypsin for 30min at RT
and additional 10min at 37oC and centrifuged at 1500g for 5 min to separate cells from
trypsinated proteoglycan fraction (16). The cell pellet was resuspended in 2M NaOH and
radioactive counts in time point samples, trypsin and cell fractions were determined using
scintillation counter.
Hyperglycemic clamping human subjects
A hyperglycemic clamp was applied for 6 hours with a target glucose concentration of 16
mmol/L (300 mg/dL) (24). To prevent hypokalemia 10mmol/L KCl was added to the glucose
solutions. Octreotide (Sandostatin ® kindly provided by Novartis, Switzerland) was dissolved
in saline and albumin and administered at final concentration of 30ng/kg/min to attenuate the
increase in endogenous insulin production in order to minimize potential confounding effects
of hyperinsulinemia (11). At this dose, octreotide has no significant vasoactive or haemostatic
side effects (24; 25) During the clamping protocol, blood glucose concentration was
measured by the glucose oxidase method (YSI 2300 STAT, Yellow Spring Inc., USA).
Target value of glucose was maintained by adjusting the infusion rate of glucose 20%
(Baxter, USA). As a time and osmolality control, the octreotide protocol was repeated on a
separate study day, during which glucose 20% was replaced with equimolar mannitol 20%
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
79
(Baxter, USA) infusion. Venous samples were obtained throughout the protocol to document
the achieved level of osmolality. Osmolality was determined by measurement of freezing
point depression on the Osmo Station (Menarini, Benelux). All samples for glucose, insulin,
and osmolality were performed in duplicate. The glutathione donor N-Acetylcysteine
(clinically graded manufactured by Department of Pharmacy, AMC) was administered as a
bolus of 100mg/kg in 15 minutes before start of glucose infusion and thereafter as a
continuous infusion of 60mg/kg throughout the identical hyperglycemia study protocol. The
rate of infusion and total amount of infused NAC in our experiment was similar to that used
for the treatment of paracetamol intoxication (18) Plasma samples were taken at time points,
0, 1, 2, 3 and 6hr during hyperglycemic clamping for hyaluronan content measurements.
Hyaluronan content
Hyaluronan mass was determined using enzyme linked immunosorbent assay kit,
commercially available from Echelon biosciences incorporated. The principle is based on
competitive ELISA assay in which the colorimetric signal is inversely proportional to the
amount of hyaluronan present in the sample.
Statistics
For statistical analysis, two-way unpaired t-tests were used. A value of P < 0.05 was
considered statistically significant. Values are means ± SE.
4.3 Results
Cummulative glucosamine and sulfate glycocalyx shedding: Effect of hyperglycemia
Figure 4.1 shows cumulative levels of glucosamine (top) and sulfate (bottom) shedded in the
media of normo- and hyperglycemic endothelial cells. Following a rapid increase in
shedding, hyperglycemic cells maintain increased levels of glucosamine shedding, 30%
higher than normoglycemic condition (32645±9773 versus 22837±630 cpm/10000cells;
P<0.05) (Figure 4.1A). Cumulative sulfate shedding shows no significant change under
hyperglycemic condition after 6 hours hyperglycemia (981±218 versus 874±181; NS) (Figure
4.1B).
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
80
Cummulative glycocalyx Glucosamine shedding
0
10000
20000
30000
40000
50000
0 100 200 300 400
T ime (min)
cpm
(10(
4)ce
lls)
NGHG
A
B Cummulative glycocalyx Sulfate shedding
0
200
400
600
800
1000
1200
1400
0 100 200 300 400
Time (min)
cpm
(10(
4)ce
lls)
NGHG
Figure 4.1: Cumulative levels of glucosamine (A) and sulfate (B) shedded in the media of normo- and
hyperglycemic endothelial cells.
Following a rapid increase in shedding, hyperglycemic cells maintain increased levels of glucosamine shedding,
30% higher than normoglycemic condition (P<0.05) (Figure 4.1A). Cumulative sulfate shedding shows no
significant change under hyperglycemic condition after 6 hours hyperglycemia (NS) (Figure 4.1B).
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
81
Glycocalyx glucosamine and sulfate content: Effect of hyperglycemia
Figure 4.2A shows an initial increase in glycocalyx glucosamine content after 1 hour of
exposure to hyperglycemia. After 6 hours, the glycocalyx glucosamine content has decreased
under hyperglycemic condition. Figure 4.2B shows an initial increase in glycocalyx sulfate
content after 1 hour of exposure to hyperglycemia. After 6 hours, the glycocalyx sulfate
content has normalized to the normoglycemic condition. After the initial increase in
glycocalyx glucosamine content under hyperglycemic condition (160±24% of
normoglycemic condition; P<0.05), after 6 hours the glycocalyx glucosamine content is
significantly decreased (81±14% of normoglycemic condition; P<0.05) (Figure 4.2C).
Figure 4.2: After an initial increase in glycocalyx sulfate content after 1 hour of exposure to hyperglycemia, at 6
hours, the glycocalyx sulfate content has normalized to the normoglycemic condition (Figure 4.2A).
glycocalyx sulfate content
0
510
1520
25
3035
40
0 100 200 300 400
Time (min)
% o
f tot
al p
ool
NGHG
A
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
82
B
C
Figure 4.2 cont: An initial increase in glycocalyx glucosamine content after 1 hour of exposure to
hyperglycemia. After 6 hours, the glycocalyx glucosamine content has decreased under hyperglycemic
condition (Figure 4.2B). After the initial increase in glycocalyx glucosamine content under hyperglycemic
condition (P<0.05), after 6 hours the glycocalyx glucosamine content is significantly decreased (P<0.05)
(Figure 4. 2C).
glycocalyx glucosamine content
0
5
10
15
20
25
30
35
40
0 100 200 300 400Time (min)
% o
f tot
al p
ool
NGHG
Effect of HG on glycocalyx content
0
50
100
150
0 100 200 300 400
Duration of hyperglycemia (min)
Glu
cosa
min
e gl
ycoc
alyx
co
nten
t (%
of N
G)
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
83
Effect of hyperglycemia on in vitro glycocalyx glucosamine shedding and human plasma
hyaluronan concentration dynamics.
Cells under hyperglycemia show an initial increase in burst in glucosamine shedding
compared to cells under normoglycemic condition (140±12%; P<0.05). After 6 hours the
glucosamine shedding is significantly higher in cells under hyperglycemia compared to
normoglycemia (140±18%; P<0.05) (Figure 4.3A). The glucosamine shedding dynamics of
cultured endothelial cells show a resemblance to the hyaluronan shedding dynamics in human
plasma of subjects exposed to 6 hours of hyperglycemic clapping (Figure 4.3B).
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
84
Figure 4.3: Cells under hyperglycemia show an initial increase in burst in glucosamine shedding compared to
cells under normoglycemic condition (P<0.05). After 6 hours the glucosamine shedding is significantly higher
in cells under hyperglycemia compared to normoglycemia (P<0.05) (Figure 4.3A). The glucosamine shedding
dynamics of cultured endothelial cells show a resemblance to the hyaluronan shedding dynamics in human
plasma of subjects exposed to 6 hours of hyperglycemic clapping, which after 6 hours of hyperglycemic
clamping the hyaluronan shedding is significantly increased. (Figure4. 3B).
A
B
Effect of HG on glycocalyx shedding
0
50
100
150
0 100 200 300 400
Duration of hyperglycemia (min)
Glu
cosa
min
e sh
eddi
ng(%
of N
G)
[HA] in plasma during acute hyperglycemia
40
60
80
100
120
140
160
180
-100 0 100 200 300 400
[HA
] in
plas
ma
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
85
4.4 Discussion
Recently, we reported on the loss of systemic glycocalyx volume in healthy volunteers during
acute exposure to 6 hours of hyperglycemia. Loss of glycocalyx volume was reflected by
significantly elevated plasma hyaluronan levels after 6 hours. In the current study, we
monitored the dynamics of glycocalyx synthesis and shedding using cultured endothelial cells
exposed to hyperglycemic conditions for 6 hours. Consistent with the clinical findings,
hyperglycemia reduced incorporation of glucosamine and sulfate into the glycocalyx of
hyperglycemic endothelial cells compared to normoglycemic paired controls. Furthermore,
the dynamics of supernatant glycocalyx shedding was remarkebly similar to the dynamics of
human plasma hyaluronan levels, showing an initial transient rise within the first hour,
followed by a more slowly, steady increase in glycocalyx shedding. Our current findings
demonstrate that the initial transient shedding increase reflects a transient hyperglycemic
increase in glycocalyx synthesis, while the later increases in glycocalyx shedding are
responsible for the net hyperglycemic loss of glycocalyx after 6 hours.
Short-term hyperglycemia has been shown to increase endothelial glycocalyx permeability
with acute capillary shutdown in C57BL/6 mice treated by normoglycemic, acutely
hyperglycemic (25 mM) for 60 min due to infusion of glucose, or hyperglycemic (25 mM)
for 2-4 wk (db/db mice). The data indicate that short-term hyperglycemia causes a rapid
decrease of the ability of the glycocalyx to exclude 70kDa dextran. No change in the vascular
permeation of 40kDa dextran was observed. These data indicate that the described increased
vascular permeability with hyperglycemia can be ascribed to an increased permeability of the
glycocalyx, identifying the glycocalyx as a potential early target of hyperglycemia (26)
Additionally studies have shown that diabetic mice display an increase in hyaluronan in
plasma compared to healthy subjects (5) and Type 2 diabetic rats show 2 fold increase in
hyaluronidase in plasma (9).
Mechanism of acute hyperglycemia induced glycocalyx damage
The mechanisms by which hyperglycemia affect hyaluronan metabolism are incompletely
understood. Brownlee (2) hypothesizes on the possibility that hyperglycemia causes diabetic
complications through an increased hexosamine pathway flux. So far, the effect of high
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
86
glucose on increased amounts of glycosaminoglycan substrates, such as glucosamine and
glucuronic acid, and its effect on glycosaminoglycan metabolism has remained undiscussed
in the literature. Since the enzyme hyaluronan synthase is situated in the cells membrane and
utilizes glucosamine and glucuronic acid precursor building blocks directly from the cytosol
without entering cellular posttranslational modification pathways, the increased substrate
pool could very well have a direct effect on the hyaluronan metabolism (17), and indirectly
stimulate hyaluronidase activity by virtue of the close association of hyaluronan synthesis and
its degradation. Additionally, studies by Wang and Hascall shows hyaluronic structures
synthesized by rat mesengial cells in response to hyperglycemia induced monocyte adhesion
(22) Studies on the effect of AGEs on vitreous of diabetic patients showed that in addition of
light exposure, AGEs promotes the decrease of MW hyaluronan (10).
Summary
Like in humans, hyperglycemia results in loss of glycocalyx on cultured cells. Loss is
secondary to an initial, transient in glycocalyx synthesis, which is reflected by a transient
supernatant peak of shedded glycocalyx constituents after 1 hour of hyperglycemia. The
initial increase in glycocalyx synthesis and shedding is followed by a maintained elevated
shedding level, resulting in a net reduction in glycocalyx content of glucosamine and sulfate.
Further studies need to determine whether activation of the glycocalyx biosynthetic pathways
by high glucose may contribute to net loss of glycocalyx protective properties during acute
exposure to hyperglycemia.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
87
References
1. Algenstaedt P, Schaefer C, Biermann T, Hamann A, Schwarzloh B, Greten H, Ruther W and Hansen-Algenstaedt N. Microvascular alterations in diabetic mice correlate with level of hyperglycemia. Diabetes 52: 542-549, 2003.
2. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813-820, 2001.
3. Capes SE, Hunt D, Malmberg K and Gerstein HC. Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: a systematic overview. Lancet 355: 773-778, 2000.
4. Ceriello A. Impaired glucose tolerance and cardiovascular disease: The possible role of post-prandial hyperglycemia. American Heart Journal 147: 803-807, 2004.
5. Chajara A, Raoudi M, Delpech B, Leroy M, Basuyau JP and Levesque H. Circulating hyaluronan and hyaluronidase are increased in diabetic rats. Diabetologia 43: 387-388, 2000.
6. Constantinescu AA, Vink H and Spaan JA. Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface. Arterioscler Thromb Vasc Biol 23: 1541-1547, 2003.
7. Esko JD, Stewart TE and Taylor WH. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 82: 3197-3201, 1985.
8. Fontijn R, Hop C, Brinkman HJ, Slater R, Westerveld A, van MJ and Pannekoek H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp Cell Res 216: 199-207, 1995.
9. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N and Takahashi T. Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats. Glycobiology 14: 65-72, 2004.
10. Katsumura C, Sugiyama T, Nakamura K, Obayashi H, Hasegawa G and Ikeda T. Effects of advanced glycation endo products on hyaluronan proteolysis: A new mechanism of diabetic vitreopathy. Ophthalmic research 36: 327-331, 2004.
11. Krentz AJ, Boyle PJ, Macdonald LM and Schade DS. Octreotide - A Long-Acting Inhibitor of Endogenous Hormone-Secretion for Human Metabolic Investigations. Metabolism-Clinical and Experimental 43: 24-31, 1994.
12. Morigi M, Angioletti S, Imberti B, Donadelli R, Micheletti G, Figliuzzi M, Remuzzi A, Zoja C and Remuzzi G. Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion. J Clin Invest 101: 1905-1915, 1998.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
88
13. Mulivor AW and Lipowsky HH. Role of glycocalyx in leukocyte-endothelial cell adhesion. Am J Physiol Heart Circ Physiol 283: H1282-H1291, 2002.
14. Nieuwdorp M, van Haeften TM, Gouverneur MCLG, Mooij HL, van Lieshout MHP, Levi M, Meijers JCM, Holleman F, Hoekstra JBL, Vink H, Kastelein JJP and Stroes ESG. Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 55: 480-486, 2006.
15. Rosenberg RD. Redesigning heparin. N Engl J Med 344: 673-675, 2001.
17. Stern R. Hyaluronan catabolism: a new metabolic pathway. European Journal of Cell Biology 83: 317-325, 2004.
18. Vale JA and Proudfoot AT. Paracetamol (Acetaminophen) Poisoning. Lancet 346: 547-552, 1995.
19. van den Berg BM, Vink H and Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res 92: 592-594, 2003.
20. Vink H and Duling BR. Capillary endothelial surface layer selectively reduces plasma solute distribution volume. American Journal of Physiology - Heart & Circulatory Physiology 278: H285-H289, 2000.
21. Vink H, Wieringa PA and Spaan JA. Evidence that cell surface charge reduction modifes capillary red cell velocity-flux relationships in hamster cremaster muscle. Journal of Physiology 489: 193-201, 1995.
22. Wang A and Hascall VC. Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion. J Biol Chem 279: 10279-10285, 2004.
23. Weinbaum S, Zhang X, Han Y, Vink H and Cowin SC. Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100: 7988-7995, 2003.
24. Williams SB, Goldfine AB, Timimi FK, Ting HH, Roddy MA, Simonson DC and Creager MA. Acute hyperglycemia attenuates endothelium-dependent vasodilation in humans in vivo. Circulation 97: 1695-1701, 1998.
25. Witzig TE, Kvols LK, Moertel CG and Bowie EJW. Effect of the Somatostatin Analog Octreotide Acetate on Hemostasis in Humans. Mayo Clinic Proceedings 66: 283-286, 1991.
26. Zuurbier CJ, Demirci C, Koeman A, Vink H and Ince C. Short-term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases lineal density of capillaries with flowing red blood cells. Journal of Applied Physiology 99: 1471-1476, 2005.
Chapter 5
Loss of Endothelial Glycocalyx during Acute
Hyperglycemia Coincides with Endothelial Dysfunction and
Coagulation Activation in vivo
Max Nieuwdorp1, Timon W. van Haeften2, Mirella C.L.G. Gouverneur3, Hans L. Mooij1,
Miriam H.P. van Lieshout1, Marcel Levi4, Joost C.M. Meijers1,
Frits Holleman4, Joost B.L. Hoekstra4, Hans Vink3,
John J.P. Kastelein1, and Erik S.G. Stroes1
Departments of Vascular Medicine1, Medical Physics3 and Internal Medicine4 , Academic
Medical Center, Amsterdam, the Netherlands
Department of Internal Medicine2, University Medical Center Utrecht, Utrecht, the
Netherlands
Diabetes. 2006 Feb;55(2):480-6.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
90
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
91
Abstract
Hyperglycemia is associated with increased susceptibility towards athero-thrombotic stimuli.
The glycocalyx, a layer of proteoglycans covering the endothelium, is involved in the
protective capacity of the vessel wall. We therefore evaluated whether hyperglycemia affects
the glycocalyx, thereby increasing vascular vulnerability. Systemic glycocalyx volume was
estimated by comparing the distribution volume of a glycocalyx permeable tracer (Dextran
40) to that of a glycocalyx impermeable tracer (labeled erythrocytes) in 10 healthy males.
Measurements were performed in randomized order on 5 occasions: 2 control measurements,
two measurements during normo-insulinemic hyperglycemia with or without N-Acetyl
cysteine (NAC) infusion, respectively, and one during mannitol infusion. Glycocalyx
measurements were reproducible (1.7 ± 0.2 versus 1.7 ± 0.3 liters). Hyperglycemia reduced
glycocalyx volume (0.8 ± 0.2 liters, p<0.05), which could be prevented by NAC (1.4 ± 0.2
liters). Mannitol infusion had no affect on glycocalyx volume (1.6 ± 0.1 liters).
t). During glucose infusion, the rate of dextran 40 plasma clearance was increased as compared with the
mannitol infusion. Depicted values on each time point are expressed as means _ SE. B: Systemic glycocalyx
volumes were determined in random order before and after infusion with glucose (bars 1 and 2) or mannitol
(bars 3 and 4) and after glucose-NAC (bar 5). Systemic glycocalyx volumes were identical at baseline;
glucose infusion resulted in a statistically significant decrease in systemic glycocalyx volume compared with
baseline, mannitol, and glucose-NAC. Data are means _ SE. *P < 0.05.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
100
Endothelial function
Flow mediated dilation showed good reproducibility between saline visits (8.8 ± 0.8 versus
8.2 ± 0.5 %, n.s; inter-session coefficient of variance of 16.8 ± 8.2%). Flow mediated dilation
was attenuated during hyperglycemia (5.8 ± 0.6 versus 8.8 ± 0.8 %, hyperglycemia vs.
baseline, p<0.05). Mannitol infusion had no effect on FMD (7.1±1.0 versus 8.2 ± 0.5%,
mannitol vs. baseline, n.s., figure 5.2). There was no difference in nitroglycerine response
after hyperglycemia vs. mannitol infusion (data not shown). Of note, due to the small sample
size, we did not determine FMD in the NAC hyperglycemia protocol.
Figure 5.2. FMD determined in a random order before and after infusion with glucose (bars 1and 2) or mannitol
(bars 3 and 4). FMD was identical before the interventions; glucose infusion resulted in a statistically significant
decrease in FMD compared with mannitol infusion and baseline. Data are means _ SE. *P < 0.05.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
101
Laboratory parameters
Plasma hyaluronan levels rose significantly during hyperglycemia (112 ± 16 versus 70 ± 6
ng/mL, HG vs. baseline p< 0.05) returning towards baseline values within 24 hours (81 ± 6
ng/mL) (figure 5.3).
Figure 5.3. Shedding of endothelial glycocalyx compounds (as assessed by plasma hyaluronan) in subjects
infused with glucose (F), mannitol (f), or glucose-NAC (OE). Data are means _ SE. *P < 0.05 vs. baseline; #P
< 0.05 among groups.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
102
Activation of the coagulation system (as indicated by an increase in F1+2 levels 1.1 ± 0.2
versus 0.4 ± 0.1 nmol/L, p<0.05) and fibrinolytic system (increase in D-Dimer levels 0.55 ±
0.2 versus 0.27 ± 0.1 g/L, p< 0.05 see figure 5.4 a and b) occurred during hyperglycemia. No
effect of mannitol was seen on these parameters. Hyperglycemia with concomitant NAC
infusion resulted in blunting of plasma hyaluronan shedding (69 ± 8 ng/mL, p<0.05, figure
5.3) and coagulation activation compared to hyperglycemia.
(F1+2: 0.8 ± 0.15 nmol/L and D-dimer: 0.44 ± 0.07 g/L,n.s. see figure 5.4a and b).
Figure 5.4. A: Activation of
coagulation system (as assessed
by prothrombin fragments 1 _ 2)
in human volunteers infused with
glucose (F), mannitol (f), or
glucose-NAC (OE). Data are
means _ SE. *P < 0.05 vs.
baseline. B: Activation of the
fibrinolytic system (as assessed by
D-dimer levels) in subjects
infused with glucose (F), mannitol
(f), or glucose- NAC (OE). Data
are means _ SE. *P < 0.05 vs.
baseline.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
103
5.4 Discussion
In the present study, we showed that the glycocalyx constitutes a large intravascular
compartment in healthy volunteers that can be estimated in a reproducible fashion in vivo.
More importantly, we showed that hyperglycemic clamping elicits a profound reduction in
glycocalyx volume that coincides with increased circulating plasma levels of glycocalyx
constituents like hyaluronan, an observation that is consistent with release of glycocalyx
constituents into the circulation. These disturbances are accompanied by impaired FMD as
well as activation of the coagulation system. Infusion of the antioxidant NAC prevented this
glycocalyx perturbation, indicating that generation of reactive oxygen species contributes to
the glycocalyx perturbation under hyperglycemic conditions.
Hyperglycemia and glycocalyx volume
Previously we validated glycocalyx measurements in isolated vessels by comparison of
erythrocyte- and Dextran 40 distribution volumes as markers of glycocalyx- impermeable and
permeable tracers, respectively (18,19). Consistent with these experimental data, we now find
comparable values for glycocalyx volume in healthy volunteers with good reproducibility of
the measurement between sessions (CV < 20%). The size of the glycocalyx volume in the
present study is in line with predictions of glycocalyx dimension in vivo, based on a thickness
of 0.5 to 3.0 µm combined with a total endothelial surface area between 1000 and 7000 m2
(27,28). After 6 h of hyperglycemic clamping, systemic glycocalyx volume is reduced to
~50% of the baseline value. This reduction coincides with a rapid increase in circulating
plasma hyaluronan levels, an important constituent of the glycocalyx. Similarly,
hyperglycemia has been associated with increased hyaluronidase activity and concomitant
increased plasma hyaluronan concentrations in animal models (17, 29). Hyaluronan has been
shown to be a principal determinant of vascular permeability, since selective removal of
hyaluronan from the vessel wall is accompanied by a profound increase in macromolecular
glycocalyx permeation (10). We presently show in vivo that loss of glycocalyx volume and
shedding of hyaluronan into the plasma is indeed accompanied by a significant increase in the
rate of Dextran 40 clearance from the circulation (figure 5.1a). This correlation between
glycocalyx volume reduction and increased permeability suggests a potential contribution of
the glycocalyx, particularly hyaluronan, to the preservation of the systemic vascular
permeability barrier.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
104
Increased formation of reactive oxygen species and glycocalyx
Several mechanisms may contribute to loss of glycocalyx volume during acute
hyperglycemia. First, hyperglycemia per se constitutes a potent pro-oxidant and pro-
inflammatory stimulus, which has been linked to enhanced degradation of the glycocalyx as
well as to shedding of hyaluronan (30). Therefore, glycocalyx loss may be secondary to a
direct effect of oxygen radicals on the synthesis of glycosaminoglycans. Indeed, in our study
infusion of the potent antioxidant NAC was able abolish the reduction in glycocalyx during
hyperglycemia. On the other hand, increased shedding of glycosaminoglycans may follow
vascular injury resulting in an upregulation of glycosaminoglycan synthesis to compensate
for stimulated increased degradation (6, 31).
Endothelial function
In conjunction with glycocalyx loss we observed a loss of flow mediated dilation after
hyperglycemic clamping. In line, several research groups have reported endothelial
dysfunction under hyperglycemic conditions (22, 32). Whereas impaired NO bioavailability
has predominantly been attributed to direct inactivation of NO by increased radical
production (33, 34), the present finding provides us with an alternative explanation. It has
been demonstrated that the endothelial glycocalyx plays an important mechano-sensory role
translating intravascular shear stress into biochemical activation of endothelial cells (12).
Accordingly, the release of NO in response to shear stress is abolished upon enzymatic
removal of glycosaminoglycans from the endothelial glycocalyx (15).
Coagulation activation
Hyperglycemia elicited coagulation and fibrinolysis, reflected by increased thrombin
generation (F1+2) as well as increased fibrinolysis (D-dimer). Our data are in line with
studies showing that induction of acute hyperglycemia in healthy volunteers increased plasma
levels of coagulation factor VIIa and stimulated tissue factor-dependent activation of
coagulation (35). The endothelial glycocalyx is a crucial compartment for binding and
regulation of enzymes involved in the coagulation cascade. In addition, the most important
inhibitor of thrombin and factor Xa, i.e. antithrombin, is firmly attached to the endothelial
glycocalyx (36). In agreement, we and others have previously demonstrated that glycocalyx
perturbation has direct effects on coagulation and fibrinolytic responses (9, 37). It is therefore
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
105
not surprising that hyperglycemia-induced loss of glycocalyx is accompanied by activation of
coagulation. The subsequent increase in endogenous fibrinolysis can thus be explained by
counterbalancing the increased thrombin generation during hyperglycemia.
Study limitations
Firstly the accuracy of glycocalyx volume estimates is determined by the accuracy of Dextran
40 distribution volume estimates. Because of its relatively small size and neutral charge,
Dextran 40 is slowly cleared from the circulation. Extrapolation of measured plasma Dextran
40 concentrations to the time of its initial intravascular injection is used to estimate
intravascular Dextran 40 concentration prior to leakage. However, as can be appreciated from
the average clearance curves in figure 1a, the error of the estimated initial Dextran 40
concentration is relatively small and will therefore have no major impact on the estimates of
glycocalyx volume. Second, the stable circulating blood volumes during hyperglycemic
clamping cannot exclude changes of microcirculatory volume. Due to anatomical dimensions
the largest part of the erythrocyte volume is located in the macrovasculature, whereas
measured dextran 40 volume is mainly situated in the microvasculature. In fact, we recently
reported that hyperglycemia reduces perfused murine capillary density up to 38% (38).
Hence, in addition to glycocalyx shedding, impaired microcirculatory perfusion may also
have contributed to the reduction of systemic glycocalyx. Finally, during hyperglycemic
clamping, an increase in insulin levels was observed in spite of the concomitant infusion of
octreotide. Higher octreotide administration was not feasible due to gastro-intestinal side
effects (23). Under these circumstances, plasma insulin increases are inextricably entangled
with hyperglycemic clamping in humans (22). Although the insulin levels were within
physiological range, we cannot exclude a potential confounding effect of insulin.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
106
Clinical implications
Experimental studies have shown that the glycocalyx constitutes a crucial intravascular
compartment, which mediates transduction of shear-stress induced NO release, modulates
vascular permeability and harbors a wide array of anticoagulant proteins. In a time course
comparable to the loss of glycocalyx volume, we find loss of shear-stress induced NO-
release, increased vascular permeability and activation of coagulation during hyperglycemic
clamping in healthy volunteers. The prevention of glycocalyx damage by antioxidant infusion
confirms other research in this area on the role of oxidative stress in hyperglycemia-induced
vascular damage (6, 39). Therefore, glycocalyx measurement may hold a promise as a tool to
estimate cardiovascular risk and the impact of cardiovascular risk lowering therapies in
patients with diabetes.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
107
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in shear induced endothelium derived nitric oxide release. Am J Phys 2003;285(2):H722-6 13. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM. Heparan eparin proteoglycan is a
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implications of perioperative red cell volume measurement with a nonradioactive marker (sodium fluorescein). Anesth Analg. 1998 ;87(6):1234-8.
21. van Kreel BK, van Beek E, Spaanderman ME, Peeters LL. A new method for plasma volume
measurements with unlabeled Dextran-70 instead of 125I-labeled albumin as an indicator. Clin Chim Acta. 1998 ;275(1):71-80.
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23. Krentz AJ, Boyle PJ, Macdonald LM, Schade DS. Octreotide: a long-acting inhibitor of endogenous
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hemostasis in humans. Mayo Clin Proc. 1991;66(3):283-6. 25. Vale JA, Proudfoot AT. Paracetamol (acetaminophen) poisoning. Lancet 1995;346:547-52. 26. Hijmering ML, Stroes ES, Pasterkamp G, Sierevogel M, Banga JD, Rabelink TJ. Variability of flow
mediated dilation: consequences for clinical application. Atherosclerosis. 2001;157(2):369-73. 27. Klitzman B and Duling BR. Microvascular hematocrit and red cell flow in resting and contracting
striated muscle. Am.J.Physiol 1979;237:H481-H490, 28. Desjardins C, Duling BR. Microvessel hematocrit: measurement and implications for capillary oxygen
transport. Am.J.Physiol 1987; 252:H494-H503 29. Wang A, Hascall VC. Hyaluronan structures synthesized by rat mesangial cells in response to
hyperglycemia induce monocyte adhesion. J Biol Chem. 2004 279:10279-85. 30. Mulivor AW, Lipowsky HH. Inflammation- and ischemia-induced shedding of venular glycocalyx. Am
J Physiol Heart Circ Physiol. 2004 ;286(5):H1672-80. 31. Henry CB, Duling BR. TNF-alpha increases entry of macromolecules into luminal endothelial cell
glycocalyx. Am J Physiol Heart Circ Physiol 2000; 279:H2815–H2823.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
endothelium-dependent vasodilation in healthy adults without diabetes: an effect prevented by vitamins C and E. J Am Coll Cardiol. 2000 ;36(7):2185-91.
33. Timimi FK, Ting HH, Haley EA, Roddy MA, Ganz P, Creager MA.Vitamin C improves endothelium-
dependent vasodilation in patients with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 1998 ;31(3):552-7
34. Cosentino F, Luscher TF. Tetrahydrobiopterin and endothelial function. Eur Heart J. 1998;19 Suppl
G:G3-8. 35. Rao AK, Chouhan V, Chen X, Sun L, Boden G: Activation of the tissue factor pathway of blood
coagulation during prolonged hyperglycemia in young healthy men. Diabetes 1999 ; 48 :1156-1161 36. Esmon CT. Inflammation and thrombosis. J Thromb Haemost 2003; 1:1343–1348 37. Pearson MJ, Lipowsky HH. Effect of fibrinogen on leukocyte margination and adhesion in
postcapillary venules. Microcirculation 2004; 11:295–306. 38. Zuurbier CJ, Demirci C, Koeman A, Vink H, Ince C. Short-term hyperglycemia increases endothelial
glycocalyx permeability and acutely decreases lineal density of capillaries with flowing RBC’s. J Appl Physiol. 2005; 99:1471-1476
39. Kurzelewski M, Czarnowska E, Beresewicz A: Superoxide- and nitricoxygen-derived species mediate
endothelial dysfunction, endothelial glycocalyx disruption, and enhanced neutrophil adhesion in the post-ischemic guinea-pig heart. J. Physiol Pharmacol. 2005; 56:163-178
110
Chapter 6
Vasculoprotective Properties of the Endothelial
Glycocalyx: Effects of Fluid Shear Stress
Mirella Gouverneur1, Bernard van den Berg2, Max Nieuwdorp3,
Erik Stroes3, & Hans Vink1
Department of Medical Physics1, Academic Medical Center, University of Amsterdam,
Amsterdam, the Netherlands, Department of
Molecular and Vascular Medicine2, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, MA, USA, and Department of Vascular
Medicine3, Academic Medical Center, University of Amsterdam, Amsterdam, the
Netherlands
J Intern Med. 2006 Apr;259(4):393-400
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
112
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
113
Abstract The endothelial glycocalyx exerts a wide array of vasculoprotective effects via inhibition of
coagulation and leukocyte adhesion, by contributing to the vascular permeability barrier and
by mediating shear stress induced NO release. In this review we will focus on the relation
between fluid shear stress and the endothelial glycocalyx. We will address the hypothesis that
modulation of glycocalyx synthesis by fluid shear stress may contribute to thinner
glycocalyces, and therefore more vulnerable endothelium, at lesion prone sites of arterial
bifurcations. Finally, we will discuss the effects of known atherogenic stimuli such as
hyperglycemia on whole body glycocalyx volume in humans and its effect on endothelial
function.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
114
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
115
6.1 Introduction
Cardiovascular disease is the major cause of mortality world wide and notwithstanding many
efforts to reduce cardiovascular disease burden, current strategies aimed at lowering systemic
risk factors have only achieved a 20-30% reduction in cardiovascular event rate (1). The
remaining 70 – 80% of events highlights the need for novel strategies to improve
cardiovascular outcome. The insight that all cardiovascular risk factors inflict loss of anti-
atherogenic properties of the vessel wall, has shifted attention from only treating systemic
risk factors towards augmenting vasculoprotective properties of the vessel wall itself. Since
the endothelium is the first line defense mechanism against atherosclerosis, much research
effort has focused at novel strategies to improve endothelial function.
Over the past several years, it is recognized that the endothelial glycocalyx may contribute to
the protection of the vascular wall against disease. The glycocalyx, consisting of a negatively
charged, organized mesh of membranous glycoproteins, proteoglycans, glycosaminoglycans
and associated plasma proteins, is situated at the luminal side of all blood vessels (2). Its
major constituents comprise hyaluronic acid (HA) and the negatively-charged eparin
sulphate proteoglycans. Glycocalyx dimensions depend upon the balance between
biosynthesis and enzymatic or shear-dependent shedding of its components (3), and whereas
historically this layer was thought to be confined to a thickness of only several nanometers, it
has recently been demonstrated to reach up to 0.5 – 3 µm intraluminally (4, 5). This relatively
large dimension of the glycocalyx, which exceeds the thickness of the endothelium and the
length of leukocyte adhesion molecules, has triggered researchers to study its role in the
course of atherogenesis (6).
Numerous studies in both micro- and macrovasculature have demonstrated that constituents
of the glycocalyx, such as hyaluronan, are intimately involved in vascular homeostasis, such
as maintaining the vascular permeability barrier (7) and regulating the release of nitric oxide
(NO) by serving as a mechano-shear sensor for NO-release (8-11). In addition, the
glycocalyx harbors a wide array of enzymes that might contribute to its vasculoprotective
effect. Thus, extracellular superoxide dismutase (ec-SOD), an enzyme which dismutates
oxygen radicals to hydrogen peroxide (12), is bound to proteoglycans within the glycocalyx.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
116
Damage to the glycocalyx is accompanied by increased shedding of ec-SOD, which results in
a dysbalance in favor of a pro-oxidant state (13). Collectively, these observations are of
particular interest since altered vascular permeability, attenuated NO-bioavailability and
redox dysregulation are amongst the earliest characteristics of atherogenesis (14). In spite of
these observations, it has proven difficult to show direct relevance of the glycocalyx as a
vasculoprotective paradigm for larger vessels. The latter is predominantly due to the fact that
glycocalyx research has traditionally focused at the microvasculature, in which atherogenesis
does not occur.
6.2 Structural properties of the endothelial glycocalyx
The first visualization of the endothelial glycocalyx was performed by conventional electron
microscopy using the cationic dye ruthenium red, which has a high affinity for acidic
mucopolysaccharides (15). Electron micrographs revealed a small irregular shaped layer
extending approximately 50- to 100 nm into the vessel lumen. Subsequent approaches with
varying perfusate contents or fixatives revealed stained structures on continuous endothelial
cell surfaces throughout diverse microvascular beds, arterial- and venular macrovessels with
large variations in dimension and appearance (4, 16-20). Fenestrated endothelium, in
addition, was found to have a combination of surface bound stained structures, about 50 –
100 nm thick, and distinct filamentous plugs composed of 20 to 40 filaments with a length of
about 350 nm on the surface of the endothelial fenestrae (21). These studies, especially when
specific approaches were applied to stabilize anionic carbohydrate structures to prevent loss-
and or collapse of these structures, gave evidence for a thick endothelial surface layer
throughout the whole vascular tree (Figure 6.1). In addition, co-localization of lectins to the
observed stained structures confirmed its saccharine nature in several of these studies (4, 16,
18).
Intravital microscopy studies on cremaster muscle showed dramatic differences between
microvascular- and systemic hematocrit (22) that could be abrogated upon enzymatic
treatment of the microvascular network with heparinase (23). From these studies a 0.3 – 1
µm thick slow-moving plasma layer on the endothelial cell surface consisting principally of
eparin-sulfate proteoglycans was thought to be involved. The first visual evidence of a 0.4
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
117
– 0.5 µm thick continuous endothelial cell surface layer was provided by comparing the
width of the flowing plasma column containing large, anionic fluorescein-labeled dextrans
with the anatomic capillary diameter as defined by the position of the luminal endothelial cell
boundaries (24). Based on observations in this study, theoretical studies predicted a
glycocalyx thickness of 0.5 – 1 µm to account for observed variations in red-cell motion
through microvessels and the discrepancy between in vivo and in vitro estimates of resistance
to blood flow (25-27). Indeed, such differences in blood flow resistance have been observed
between control- and hyaluronidase treated vessels in a study of coronary reactive hyperemia
in a dog (28). Enzymatic degradation of the glycocalyx with hyaluronidase has been shown
to significantly increase the available intralumenal space for flowing blood (7).
2 µm2 µm
EndothelialEndothelialEndothelialcellcellcell
GlycocalyxGlycocalyxGlycocalyx
0.2 µm0.2 µm2 µm2 µm
EndothelialEndothelialEndothelialcellcellcell
GlycocalyxGlycocalyxGlycocalyx
EndothelialEndothelialEndothelialcellcellcell
GlycocalyxGlycocalyxGlycocalyx
0.2 µm0.2 µm
Figure 6.1a: electron micrographs of goat capillary glycocalyx (Courtesy of Dr. Bernard van den Berg)
Lumen
0.5 µm
EC
Lumen
0.5 µm
EC
Figure 6.1b: examples of the spatial heterogeneity of glycocalyx dimensions in the vascular system (Courtesy of Dr. Bernard van den Berg).
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
118
Although various studies are consistent with the concept that perturbation of the glycocalyx
contributes to increases in endothelial vulnerability upon ischemia/reperfusion (17), hypoxia
(20), exposure to low-density lipoproteins (29, 30) and atherogenic shear stress profiles (6,
18), it has proven difficult to show direct relevance of the glycocalyx as a vasculoprotective
paradigm for larger vessels. The latter is predominantly due to the fact that glycocalyx
research has traditionally focused at the microvasculature, in which atherogenesis does not
occur. However, several studies have emphasized that the relevance of the glycocalyx is not
confined to smaller vessels (6, 17). For example, van Haaren et al recently visualized a thick
endothelial glycocalyx in larger arteries in rats (5). The glycocalyx in larger vessels has also
been shown to decrease extravasation of LDL particles into the subendothelial space (31, 32).
Amongst others, these data imply that also in the macrovasculature the glycocalyx adds
towards the vasculoprotective properties of the vessel wall.
6.3 Glycocalyx at arterial bifurcations
Although reduced levels of surface bound sialic acids (33) and increased endothelial
permeability and susceptibility to atherosclerotic lesion formation (18) have been found to
coincide with arterial branch points and curvatures, little is known about the contribution of
glycocalyx perturbation to the increased vascular vulnerability of high atherogenic risk areas.
Atherosclerotic lesions within the arterial tree develop at predictable vessel geometries, e.g.
arterial branching and curvatures, and constraints on vessel motion by the surrounding
tissues, which lead to local flow instabilities and separations. Such lesions can be detected
and visualized as changes in vascular wall properties and quantified as intima media ratios
(IMR). Increases in IMR have been found to be associated with increased cardiovascular risk
factors and atherosclerosis (37, 38, 39).
In a recent study, van den Berg et al. (6) hypothesized that endothelial cells, which play a
central role in response to shear stress (40), express a modified surface glycocalyx at high
atherogenic risk regions and, in turn, contribute to predisposition of these arterial sites to
atherosclerotic lesion formation. The endothelial glycocalyx dimension was investigated by
electron microscopic observation at low- and high risk regions of the C57Bl/6J mouse carotid
artery, using the common- and internal carotid bifurcation (sinus) area as a model for arterial
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
119
sites exposed to low- and high atherogenic risk, respectively (41). As shown in Figure 6.2, it
is clear that the dimension of the endothelial glycocalyx at the sinus region of the mouse
internal carotid artery is significantly smaller than the glycocalyx dimension on the luminal
surface of the common carotid artery. This finding is in support of the hypothesis that
perturbation of the glycocalyx contributes to the increased vascular vulnerability of regions
that are at high atherogenic risk. Furthermore, this thinner glycocalyx is accompanied by
greater intima media ratios and a thicker subendothelial layer, indeed confirming that
regional differences in glycocalyx dimension reflect variations in its vasculo-protective
capacity.
0
100
200
300
400
500
600
C57Bl6 ApoE*3 C57Bl6 ApoE*3
Gly
coca
lyx
thic
knes
s (n
m)
Common carotidregion
Internal carotid sinusregion
* * *
Figure 6.2a: Glycocalyx dimension is diminished at the atheroprone sinus region of the internal carotid
artery in mice compared to the atheroprotected common carotid artery. Systemic atherogenic stimulation by
a hyperlipidemic, hypercholesterolemic diet for 6 weeks in ApoE3-Leiden mice further diminishes the
dimension of the glycocalyx in the common carotid artery (From reference 6).
* = P < 0.05 compared to common region of C57Bl6 mice on normal diet.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
120
0
200
400
600
800
1000
C57Bl6 ApoE*3 C57Bl6 ApoE*3
Sube
ndot
helia
l mat
rix th
ickn
ess
(nm
)
Common carotidregion
Internal carotid sinusregion
*
*
*C
Figure 6.2b: Greater dimensions of the subendothelial intima layer result in greater intima-to-media ratios (IMR) at
vulnerable sites of the carotid arterial bifurcation with diminished glycocalyx dimensions (From reference 6).
* = P < 0.05 compared to common region of C57Bl6 mice on normal diet..
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
121
Previous studies have demonstrated that loss of glycosaminoglycans from the endothelial
glycocalyx by enzyme treatment is associated by edema formation of the subendothelial
space (4), indicating that flow profile related modulation of the glycocalyx might contribute
to the earlier observed progression from a decreased endothelial barrier function into
subsequent intimal edema at vascular regions exposed to disturbed flow (42). Whether
edema formation contributed to the increased IMR in the present study remains to be
explored. However, the site specific differences in IMR occurred in the absence of changes
in the dimension of the media layer, and were predominantly due to increases in the
dimension of the subendothelial space. Furthermore, no evidence was found for
accumulation of blood cells or monocytes in the intima layer, indicating that the contribution
of the inflammatory response was minimal at this stage.
6.4 Mechanism of glycocalyx reduction at high risk regions
The fact that the glycocalyx dimension is significantly diminished at the sinus region
compared to the glycocalyx dimension at the opposite site of the internal carotid near the
flow divider as well as at the common carotid area just proximal to the carotid bifurcation,
suggests that spatial differences in glycocalyx dimension are related to local variations in
flow profiles. It is well known that areas of high atherogenic risk are located close to regions
of disturbed flow at arterial bifurcations. Therefore, it is tempting to speculate that
undisturbed flow patterns and the associated stimulation of vascular endothelium by fluid
shear stress are essential to obtain optimal glycocalyx protective properties. However,
although studies have recently demonstrated that the endothelial glycocalyx indeed plays an
important role in mechanotransduction of fluid shear stress, very few data are available on the
relation between fluid shear stress and glycocalyx synthesis.
Earlier studies, using sialic acid binding lectins (33) and alcian blue (18), showed that
reduced dimensions of the endothelial glycocalyx at arterial sites exposed to disturbed flow
patterns associate with increases in endothelial permeability and susceptibility to
atherosclerotic lesion formation. Additionally, studies by Woolf (43) and Wang et al. (44)
revealed thicker glycocalyces at high shear regions compared to low shear regions and
demonstrated that glycocalyx dimension is reduced when rabbits are fed an atherogenic diet.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
122
Steady state glycocalyx dimension is the result of local synthesis and degradation of its
constituents and it is important to know the factors that determine this balance.
Recently, Gouverneur (45) demonstrated that exposure of cultured endothelial cells for 24 h
to a shear stress of 10 dynes/cm2 stimulates incorporation of glucosamine-containing
glycosaminoglycans (GAGs) in the glycocalyx, which is accompanied by elevated levels of
glucosamine-containing GAGs in the supernatant. These increases were confirmed by direct
demonstration of increased hyaluronan concentrations in the glycocalyx and in the
supernatant, as well as by a 3 fold increase in the incorporation of hyaluronan binding protein
in the glycocalyx. In addition to its incorporation in hyaluronan, glucosamine is also
incorporated in sulfated sugars like eparin sulfate and chondroitin sulfate. In addition,
Arisaka et al.(46) used pig aortic endothelial cells exposed to shear stress levels of 15 and 40
dynes/cm2 in a parallel flow chamber for periods of 3, 6, 12 and 24 h. These authors
demonstrated increased synthesis of sulfated GAGs after high shear stress of 40 dynes/cm2,
and also a small, but significant increase at 15 dynes/cm2. Similarly, Elhadj et al (47) exposed
bovine aortic endothelial cells for 7 days to < 0.5 dynes/cm2 prior to increasing shear rates for
3 days to 5 and 23 dynes/cm2. No significant increase in the net sulfated GAG synthesis was
detected, but a shift in its size distribution was reported, indicating that modulation of specific
sulfation patterns may occur despite limited effects on sulfated GAG synthesis. In summary,
these experiments demonstrate that shear stress increases hyaluronan content in the
endothelial glycocalyx, that shear stress exposure alters the size distribution of endothelial
sulfated GAGs, and that high levels of shear stress may also increase sulfated GAG synthesis.
6.5 Glycocalyx and systemic atherogenic stimuli
In addition to the spatial differences in glycocalyx dimension at arterial bifurcations, van den
Berg et al. (6) also reported that the glycocalyx is diminished upon systemic atherogenic
challenge by a high fat- high cholesterol diet. Systemic perturbation of the glycocalyx by
hypercholesterolemia and/or hypertriglyceremia on top of pre-existing regional variations in
glycocalyx protective properties, introduced further increases in vascular vulnerability. The
mechanism by which the glycocalyx is diminished in atherogenic mice remains to be
elucidated, but the present finding is consistent with previous studies demonstrating rapid
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
123
shedding of glycocalyx from the endothelial surface upon acute stimulation with elevated
plasma levels of Ox-LDL or by acute exposure of the endothelium to inflammatory agents
like thrombin or TNF-α (48, 49, 50). In conclusion, both regional and risk factor induced
increases in atherogenic risk are associated by smaller glycocalyx dimensions and greater
IMR. Exposure of the high risk sinus area to an additional atherogenic challenge results in
endothelial thickening and excessive swelling of the subendothelial space, in line with the
proposed hypothesis that vascular sites with diminished glycocalyx are more vulnerable to
pro-inflammatory and atherosclerotic sequelae.
6.6 Human glycocalyx measurements
To date, direct visualization of endothelial glycocalyx in humans has been unsuccessful,
mainly due to the fact that the endothelial glycocalyx is a very delicate structure depending
critically on the presence of flowing plasma (2). As a consequence, the best way to measure
the endothelial glycocalyx in humans is to compare systemic intravascular distribution
volumes for glycocalyx permeable versus glycocalyx impermeable tracers. Subtracting these
two volumes provides an estimate of whole body glycocalyx volume (51).
At present, Nieuwdorp et al. (51) try to answer the question whether glycocalyx perturbation
mediated vascular vulnerability contributes to the accelerated rate of atherogenesis in patients
with type 1 diabetes. Whereas this is at least in part the consequence of increased prevalence
of traditional cardiovascular risk factors, these cannot fully explain the propensity towards
cardiovascular complications in diabetic patients (52). Disease-specific abnormalities, such as
hyperglycemia, may also facilitate the development of vascular lesions in these patients.
Thus, hyperglycemia has been shown to induce a wide array of downstream effects, which
may adversely affect the protective capacity of the vessel wall (53). Since increased
degradation of proteoglycans has indeed been demonstrated in hyperglycemic conditions (54,
55), the impact of hyperglycemia on the glycocalyx merits special interest. Therefore,
Nieuwdorp et al. recently set out to evaluate the impact of hyperglycemia on the glycocalyx
in healthy volunteers. Systemic glycocalyx volume was measured before and 6 hours after
normo-insulinemic, hyperglycemic clamping.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
124
Interestingly, Nieuwdorp et al. demonstrate that the glycocalyx constitutes a large
intravascular compartment of up to 2 liters in healthy volunteers (Figure 6.3), which can be
estimated in a reproducible fashion. More importantly, they show that hyperglycemic
clamping elicits a profound reduction in glycocalyx volume coinciding with increased
circulating plasma levels of glycocalyx constituents like hyaluronan, consistent with release
of glycocalyx constituents into the circulation upon hyperglycemia. These disturbances are
accompanied by impaired flow mediated dilation as well as activation of the coagulation
system. Taken in conjunction with available experimental data, the present findings imply
that glycocalyx perturbation may be a novel mechanism contributing to enhanced
vulnerability of the vessel wall under hyperglycemic conditions. Similarly, several other
research groups have reported endothelial dysfunction under hyperglycemic conditions (56,
57). Whereas impaired NO bioavailability has predominantly been adjudicated to direct
inactivation of NO by increased radical production (58, 59), the present finding provides us
with an alternative option. It has been acknowledged that the glycocalyx serves as part of the
endothelial mechanosensor, which translates intravascular shear stress into biochemical
activation of endothelial cells (9-11, 60). Accordingly, the release of NO by endothelial cells
in response to shear stress is abolished upon enzymatic removal of glycosaminoglycans from
the endothelial glycocalyx (9, 10, 60). It is tempting to speculate that loss of glycocalyx may
have contributed to the impaired shear-mediated NO release during hyperglycemia.
Effect of Hyperglycemia on Human Glycocalyx Volume
0
1
2
Control Hyperglycemia
Gly
coca
lyx
volu
me
(lite
rs)
P < 0.05
Effect of Hyperglycemia on Human Glycocalyx Volume
0
1
2
Control Hyperglycemia
Gly
coca
lyx
volu
me
(lite
rs)
P < 0.05Figure 6.3: Effect of 6h acute
hyperglycemia (16 mmol/l) on
systemic glycocalyx volume in
healthy human volunteers
(Reproduced from reference 51).
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
125
6.7 Summary
Currently available evidence in animal models shows that the glycocalyx exerts a wide array
of anti-atherogenic effects via inhibition of coagulation and leukocyte adhesion, by
contributing to the vascular permeability barrier as well as by mediating shear stress induced
NO release. In agreement with the hypothesis that glycocalyx perturbation increases
endothelial vulnerability, the dimension of the endothelial glycocalyx at atherogenic lesion
prone sites is significantly smaller than its dimension on the luminal surface of the
atheroprotected common carotid artery. Furthermore, focal sites with diminished glycocalyx
dimension appear to be more sensitive to further provocation by systemic atherogenic stimuli.
Most intriguing is the finding that relatively great systemic glycocalyx volumes in healthy
volunteers are significantly reduced upon exposure to atherogenic risk factors. As yet, this
finding does not prove causality of glycocalyx derangement in mediating elevated
atherogenic risk and future studies need therefore to address whether restoration of the
glycocalyx in itself is able to slow down or even reverse the progression of atherosclerotic
disease. Nevertheless, systemic glycocalyx measurement may hold a promise as a diagnostic
tool to estimate cardiovascular risk as well as to evaluate the impact of cardiovascular risk
lowering or even glycocalyx restoring therapeutic interventions.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
126
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