Original Article

Endothelial Glycocalyx Damage Coincides With

Microalbuminuria in Type 1 Diabetes
Max Nieuwdorp,1 Hans L. Mooij,1 Jojanneke Kroon,1 Bektas Atasever,2 Jos A.E. Spaan,3 Can Ince,2
Frits Holleman,4 Michaela Diamant,5 Robert J. Heine,5 Joost B.L. Hoekstra,4 John J.P. Kastelein,1
Erik S.G. Stroes,1 and Hans Vink3

Chronic hyperglycemia underlies microvascular complications in patients with type 1 diabetes. The mechanisms
leading to these vascular complications are not fully understood. Recently, we observed that acute hyperglycemia
results in endothelial glycocalyx damage. To establish
whether glycocalyx is associated with microvascular damage, we performed glycocalyx perturbation volume measurements in type 1 diabetic patients with microalbuminuria
(DM1-MA group; n 7), without microalbuminuria (DM1-NA
group; n 7), and in age-matched control subjects (CON;
n 7). Systemic glycocalyx volume was determined comparing intravascular distribution volume of a glycocalyxpermeable tracer (dextran 40) to that of a glycocalyximpermeable tracer (labeled erythrocytes). Sublingual
capillaries were visualized using orthogonal polarization
spectral microscopy to estimate microvascular glycocalyx.
Patients and control subjects were matched according to
age and BMI. Glycocalyx volume decreased in a stepwise
fashion from CON, DM1-NA, and finally DM1-MA subjects
(1.5 0.1, 0.8 0.4, and 0.2 0.1 l, respectively, P < 0.05).
Microvascular glycocalyx in sublingual capillaries was also
decreased in type 1 diabetes versus the control group
(0.5 0.1 vs. 0.9 0.1 m, P < 0.05). Plasma hyaluronan,
a principal glycocalyx constituent, and hyaluronidase were
increased in type 1 diabetes. In conclusion, type 1 diabetic
patients are characterized by endothelial glycocalyx damage, the severity of which is increased in presence of
microalbuminuria. Diabetes 55:11271132, 2006

ype 1 diabetesassociated micro- and macrovascular complications are major causes of morbidity and mortality (1,2). In this disorder, the
presence of microvascular disease is strongly
associated with increased cardiovascular risk, underlining
the generalized nature of such vascular dysfunction (3,4).

From the 1Department of Vascular Medicine, Academic Medical Center,

Amsterdam, the Netherlands; the 2Department of Clinical Physiology, Academic Medical Center, Amsterdam, the Netherlands; the 3Department of
Medical Physics, Academic Medical Center, Amsterdam, the Netherlands; the
4
Department of Internal Medicine, Academic Medical Center, Amsterdam, the
Netherlands; and the 5Department of Endocrinology, Diabetes Center, VU
University Medical Center, Amsterdam, the Netherlands.
Address correspondence and reprint requests to Erik Stroes, MD, PhD,
Department of Vascular Medicine, Room F4.275, Academic Medical Center
University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, Netherlands.
E-mail: [email protected].
Received for publication 14 December 2005 and accepted in revised form 9
January 2006.
OPS, orthogonal polarization spectral.
2006 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.

DIABETES, VOL. 55, APRIL 2006

The exact pathways leading to this propensity for vascular

disease have not been fully elucidated. In line with direct
adverse effects of hyperglycemia itself (5), good glycemic
control has been associated with decreased microvascular
disease rates (6,7). Whereas the concept of generalized
vascular dysfunctional state in type 1 diabetes has already
been put forward 2 decades ago (8), a final common
pathway for the vascular dysfunction has remained a
matter of debate.
Recently, we demonstrated that the endothelial glycocalyx, an intraluminal layer consisting of glycosaminoglycans and hyaluronan, constitutes an important component
to the vascular permeability barrier by preventing transvascular leakage of macromolecules (9). Conversely, hyaluronidase exposure severely decreases endothelial
glycocalyx thickness with a concomitant increase in capillary wall permeability and induction of pericapillary
edema (10,11). In fact, loss of glycocalyx leads to a wide
spectrum of vascular abnormalities in experimental models, including increased vascular permeability, adhesion of
mononuclear cells, and platelets to the endothelial surface
and attenuated nitric oxide availability (12). Restoration of
the glycocalyx is associated with reversal of these derangements (11,13). Collectively, these data suppose a
potential role for the glycocalyx in determining the protective properties of the vessel wall. Recently, we validated a technique to assess the volume of the endothelial
glycocalyx in humans. Using this technique, we found that
acute hyperglycemia resulted in a profound perturbation
of the glycocalyx, which closely coincided with vascular
dysfunction and coagulation activation (14).
In the present study, we set out to evaluate whether
glycocalyx loss is also present in patients with longstanding type 1 diabetes and whether more severe glycocalyx perturbation coincides with the presence of
microalbuminuria. To substantiate this, we determined
systemic glycocalyx volume as well as microvascular
glycocalyx thickness in type 1 diabetic patients with and
without microalbuminuria as well as in matched normoglycemic healthy control subjects.
RESEARCH DESIGN AND METHODS
We enrolled nonsmoking male type 1 diabetic patients either with (DM1-MA
group) or without (DM1-NA group) microalbuminuria, defined as albumin-tocreatinin ratio 2.5 mg/mmol in a morning urine sample. Of note, both the
DM1-MA and DM1-NA patients had been diagnosed with type 1 diabetes for at
least 15 years, with comparable mean duration of diabetes in both groups. All
patients were C-peptide negative, used multiple injections of insulin per day,
and had HbA1c (A1C) levels between 7.0 and 9.0% during the 6 months
preceding the study. The presence of macrovascular disease, defined as
electrocardiogram abnormalities, abnormal ankle-brachial index, or a history
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GLYCOCALYX PERTURBATION IN TYPE 1 DIABETES

of cardiac, cerebral, or peripheral vascular events were exclusion criteria for

the study. Sex- and age-matched, normoglycemic healthy control subjects
(CON; n 7) were also included. All subjects gave written informed consent,
and approval was obtained from the internal review board of the Academic
Medical Center. The study was carried out in accordance with the principles
of the Declaration of Helsinki.
All experiments were performed after an overnight fast in a quiet and
air-conditioned room (temperature 2224C). Participants were asked to
refrain from heavy physical exercise in the 24 h before the study visit. Patients
using ACE inhibitors or angiotensin II antagonists were asked to stop this
medication at least 5 days before the study visit. Patients with type 1 diabetes
were asked to check their fasting blood glucose level (target levels 7.0 12.0
mmol/l), since injection of insulin was not allowed from 8 h prior until the end
of the experiment. Based on 24-h urine microalbuminuria measurements in
the 6 months preceding the study, patients were selected as normalbuminuric
or microalbuminuric. At the beginning of the study, morning urine samples
were collected to verify albumin-to-creatinin ratios. Blood pressure was
measured three times, and the last two measurements were used to calculate
systolic and diastolic blood pressure. During the study, two cannulas were
inserted in the antecubital veins of both forearms for the collection of blood
and infusion of Dextran 40 and labeled autologous erythrocytes, respectively.
Laboratory methods. After centrifugation, aliquots were snap frozen and
stored at 80C. Hematocrit (Hsys) was measured after centrifugation of
heparinized blood at 10,000 rpm for 5 min (Hettich, Tuttlingen, Germany).
Total cholesterol, HDL cholesterol, and triglycerides were measured by
standard enzymatic methods (Roche Diagnostics, Basel, Switzerland). LDL
cholesterol was calculated using the Friedewald formula. Alanine aminotransferase and aspartate aminotransferase were measured by pyridoxalphosphate
activation assay (Roche Diagnostics). Creatinin was measured by Jaffe kinetic
colorimetric test (Roche Diagnostics) on Modular P800 (Roche Diagnostics).
A1C was measured by high-performance liquid chromatography (Reagens
Bio-Rad Laboratories, Veenendaal, the Netherlands) on a Variant II (Bio-Rad
Laboratories). Quantitative total plasma hyaluronan levels were measured by
enzyme-linked immunosorbent assay (Echelon Biosciences, Salt Lake City,
UT). Plasma hyaluronidase levels were determined with a previously described assay (intersample coefficient of variation 20%) (15). Urinary creatinin and albumin content was determined according to the Jaffe method on the
P800 and by immunoturbidimetric assay, respectively (Roche Diagnostics).
Estimation of systemic glycocalyx volume. The glycocalyx allows limited
access to plasma macromolecules and erythrocytes (16,17). Hence, systemic
glycocalyx volume can be estimated by comparing circulating blood volume
with the intravascular distribution volume of a glycocalyx-permeable tracer
such as neutral dextran 40 (molecular weight 40 kDa). We recently showed
that systemic glycocalyx volume can be reproducibly measured (intermeasurement coefficient of variation 15.2 9.8%) and has a relatively large
magnitude in healthy male volunteers (14). In short, the intravascular distribution volume of labeled, autologous erythrocytes was used to quantify
circulating blood volume (18). Blood was drawn and centrifuged at 1,330 rpm
for 5 min. Subsequently, 250 mg/ml of sodium fluorescein was added to the
erythrocyte fraction for 5 min. After washing, labeled erythrocytes were
resuspended in saline to the initial volume and reinfused. Subsequently, blood
was drawn at 4, 5, 6, and 7 min after infusion. The fraction of labeled
erythrocytes compared with total erythrocyte pool was used to estimate
circulating erythrocyte volume. Preinjection unlabeled erythrocytes (t 1)
served as negative controls. Labeled erythrocytes were measured using a
FACScan (FACSCalibur; Becton Dickinson, Mountain View, CA), during
which at least 100,000 cells were counted to measure the circulating fraction
of labeled erythrocytes. Circulating plasma volume was calculated from
circulating erythrocyte volume (Vrbc) and large vessel hematocrit (Hsys) by
the following formula: circulating plasma volume ([1 Hsys] Vrbc)/Hsys
(18).
Dextran 40 is used as a probe to estimate total intravascular volume, which
includes the glycocalyx compartment (14,16,17,19). A bolus of 10 ml dextran
1 (Promiten; NPBI International, Emmercompascuum, the Netherlands) was
injected to attenuate the risk for anaphylactic reactions. At least 1 h later, 100
ml dextran 40 (Rheomacrodex; NPBI International) was intravenously injected, followed by repeated blood sampling at 5, 7, 10, 15, 20, and 30 min.
Dextran 40 concentration was calculated by measuring the increase in glucose
concentration in the postinfusion samples after hydrolyzation of dextran 40
glucose polymers (19). Glucose concentration per time point was assessed in
duplicate using the hexokinase method (Gluco-quant, Hitachi 917; Hitachi). To
determine the initial intravascular distribution volume of dextran 40, the
concentration of dextran 40 at the time of injection was estimated by
exponential fitting of the measured dextran 40 concentrations.
Visualization of the capillary endothelial glycocalyx. To determine the
thickness of the endothelial glycocalyx in individual capillary blood vessels,
we used orthogonal polarization spectral (OPS) imaging of the sublingual
1128

microcirculation (Cytoscan; Cytometrics, Philadelphia, PA), which has been

extensively validated (20,21). Preceding the systemic glycocalyx volume
measurement, images were obtained with a times-five objective (on screen
magnification 650). A total of five representative sublingual capillaries per
person (n 21; 7 healthy control and 14 type 1 diabetic subjects) were
identified for microvascular glycocalyx analysis. As previously reported, the
change in capillary red cell column width following capillary leukocyte
passage can be used to provide an estimate of the anatomic capillary diameter
(i.e., glycocalyx compressed; Dcap_anat), whereas the red cell column width
before leukocyte passage reflects the functionally perfused capillary diameter
(Dcap_func) (22). Subsequently, subtracting functional capillary diameter
from anatomic capillary diameter ([Dcap_anat Dcap_func]/2) provides an
estimate of individual capillary glycocalyx thickness. Measurements and
analysis of the images were performed with Image Pro Plus (Mediacybernetrics, Silver Spring, MD) by the same person, who was unaware of the
clinical details of the participants.
Statistical analysis. All results are expressed as means SE except those
listed in Table 1 (means SD). Differences between groups were tested by
Kruskall-Wallis test. Mann-Whitney U test (two tailed) was used for comparison of vascular volume compartment determinants and unpaired Students t
test (two tailed) for all other parameters. Statistical differences were first
calculated for all type 1 diabetic patients versus control subjects and then
separately for DM1-NA versus DM1-MA subjects. Correlation coefficients
between systemic glycocalyx volume and microvascular glycocalyx thickness
and biochemical parameters were calculated with the Spearmans rank
correlation test (two tailed). A probability value of 0.05 was considered
significant.

RESULTS

Baseline parameters. Clinical characteristics of subjects

are listed in Table 1. Compared with healthy control
subjects, type 1 diabetic patients had higher fasting plasma
glucose and A1C levels (Table 1). Between DM1-NA and
DM1-MA patients, albumin-to-creatinin ratio and plasma
creatinin were significantly different (Table 1).
Systemic glycocalyx volume. All procedures during the
test day were well tolerated, and no adverse events
occurred during systemic glycocalyx volume measurements. Systemic glycocalyx volumes were significantly
decreased in type 1 diabetic patients compared with
control subjects (CON: 1.5 0.1 vs. type 1 diabetes: 0.5
0.1 l, P 0.01, Fig. 1A). Markedly, the reduction in
systemic glycocalyx volume was significantly higher in
DM1-MA compared with DM1-NA subjects (0.2 0.1 and
0.8 0.4 l, respectively, P 0.05, Fig. 1A). Changes in
dextran 40 distribution volumes were predominantly responsible for the decreased systemic glycocalyx volume in
type 1 diabetes (CON: 4.5 0.7 vs. DM1-NA: 3.7 0.9 and
DM1-MA: 3.4 0.6 l), as evidenced by the changed dextran
40 clearance curves (Fig. 1B). Circulating plasma volumes
were not significantly different (CON: 3.0 0.4 vs. DM1NA: 2.9 0.4 and DM1-MA: 3.2 0.5 l, NS). Hematocrit
values were comparable between groups and did not
change during dextran 40 infusion (data not shown).
Microvascular glycocalyx volume. Glycocalyx thickness in sublingual capillaries was reduced in type 1
diabetic patients compared with control subjects (CON:
0.9 0.1 vs. type 1 diabetes: 0.5 0.1 m, P 0.01), with
a nonsignificant difference between DM1-NA and DM1-MA
subjects (0.5 0.1 and 0.4 0.1 m, respectively, Fig. 2A).
The reduced glycocalyx thickness in type 1 diabetic patients was accompanied by a modest reduction in anatomic capillary diameter compared with control subjects
(CON: 6.8 0.2 vs. DM1-NA: 5.7 0.1 and DM1-MA: 5.0
0.2 m, P 0.05). In addition, a close correlation was
found between systemic glycocalyx volume and sublingual
glycocalyx thickness in all subjects (r 0.73, P 0.01, Fig.
2B).
DIABETES, VOL. 55, APRIL 2006

M. NIEUWDORP AND ASSOCIATES

TABLE 1
Clinical characteristics of the study subjects

n
Duration of diabetes (years)
Daily insulin use (IU)
Age (years)
BMI (kg/m2)
Smoking (yes/no)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Heart rate (bpm)
Fasting plasma glucose (mmol/l)
Total cholesterol (mmol/l)
LDL cholesterol (mmol/l)
HDL cholesterol (mmol/l)
Triglycerides (mmol/l)
Albumin-to-creatinine ratio (mg/mmol)
Plasma creatinine (mol/l)
Aspartate aminotransferase (U/l)
Alanine aminotransferase (U/l)
A1C (%)
High-sensitivity C-reactive protein (mg/l)

CON group

DM1-NA
group

DM1-MA
group

52 11
24 4
0/7
134 18
74 10
64 7
5.1 0.4
4.8 0.8
3.0 0.6
1.5 0.3
0.6 0.2
0.4 0.1
69 6
21.9 5.9
22.0 11.6
5.2 0.3
1.3 1.1

7
29 13
50 18
47 12
23 2
0/7
137 10
77 7
64 10
9.1 2.4*
4.8 1.0
2.9 0.9
1.6 0.4
0.6 0.2
0.8 0.7
67 6
23.9 4.6
22.6 8.1
7.7 0.6*
1.6 1.2

7
32 7
48 17
51 10
24 3
0/7
150 17
80 9
70 10
9.3 2.2*
4.8 0.8
2.6 0.8
1.9 0.4
0.6 0.2
30 27*
86 1*
24.0 5.0
19.3 5.1
8.3 1.0*
2.8 1.6

Data are means SD. *P 0.05 control vs. diabetic subjects. P 0.05 DM1-NA vs. DM1-MA subjects.

Systemic biochemical markers of glycocalyx perturbation. Plasma levels of hyaluronan were increased in
type 1 diabetes (DM1: 118 9 vs. CON: 65 8 ng/ml, P
0.01), with a further increase in type 1 diabetes with
microalbuminuria (DM1-MA: 136 29 vs. DM1-NA: 100
17 ng/ml, P 0.05, Fig. 3A). Plasma hyaluronidase levels
were increased in type 1 diabetic patients (type 1 diabetes:
236 8 vs. CON: 170 19 units/ml, P 0.01), with a trend
toward higher values in type 1 diabetes with microalbuminuria (DM1-MA: 240 13 and DM1-NA: 232 10
units/ml, Fig. 3B). Plasma hyaluronan and hyaluronidase
(r 0.75 and 0.66, respectively, P 0.01), plasma
creatinin (r 0.58, P 0.05), and albumin-to-creatinin
ratio (r 0.54, P 0.05) were inversely correlated with
systemic glycocalyx volume. Systemic glycocalyx volume
showed no correlation with fasting plasma glucose (r
0.31, NS), but there was a trend for A1C (r 0.41, P
0.06).
DISCUSSION

In the present study, we show that systemic glycocalyx

volume is markedly reduced in patients with long-standing
type 1 diabetes compared with normoglycemic control
subjects. In fact, the magnitude of systemic glycocalyx
reduction was largest in type 1 diabetic patients with
microalbuminuria. Using OPS imaging of the sublingual
microcirculation, we were also able to confirm the reduction of glycocalyx volume in type 1 diabetic patients at the
level of the microcirculation. The reduction of systemic
glycocalyx volume was correlated with increased levels of
circulating hyaluronan and its degrading enzyme hyaluronidase. The close relation between glycocalyx perturbation
and microvascular complications in type 1 diabetes warrants further studies to assess whether loss of glycocalyx
may actually be a causal factor for vascular complications
in type 1 diabetes.
Systemic and microvascular glycocalyx reduction in
patients with type 1 diabetes. Previously, we have
validated the estimation of glycocalyx volume by comparDIABETES, VOL. 55, APRIL 2006

ison of erythrocyte and dextran 40 distribution volumes in

individual vessels and in vivo (14,16,17). The size of
systemic glycocalyx volume in healthy control subjects is
in line with its predicted dimensions, based on a thickness
between 0.5 and 3.0 m (23,24) and an estimated endothelial surface area between 1,000 and 7,000 m2 (25). Microvascular OPS imaging of glycocalyx thickness in
sublingual capillaries further substantiated our systemic
findings. In analogy with our previous findings of glycocalyx perturbation during acute hyperglycemia in healthy
individuals (14), a marked reduction in systemic glycocalyx volume was observed in type 1 diabetic patients. In
fact, overall systemic glycocalyx volume was reduced to
40 50% of the volume observed in matched normoglycemic control subjects, with an even more profound reduction in type 1 diabetic patients characterized by
microalbuminuria. In addition, we also found a trend
between A1C levels and glycocalyx volume, whereas no
correlation was found for fasting plasma glucose levels.
Therefore, the impact of (long-term) glucose regulation on
glycocalyx volume will have to be addressed in a larger
cohort of type 1 diabetic patients.
Interestingly, the reductions in dextran 40 intravascular
distribution volumes in type 1 diabetic patients are not
accompanied by concomitant increases in circulating
blood volume. The latter implies that the lost glycocalyx
volume is apparently compensated for by a reduction in
total vascular volume. Since the majority of the glycocalyx
volume is located within the microvasculature, these
changes should become apparent at the level of the
capillaries. Indeed, we observed a significant reduction in
anatomic capillary dimensions in type 1 diabetes. The
mechanism by which glycocalyx loss leads to a decrease in
capillary diameter needs further study. One of the mechanisms could relate to the formation of pericapillary
edema, which has been shown to cause a reduction of
capillary diameters following glycocalyx degradation upon
hyaluronidase infusion in rats (10). In analogy, reductions
in capillary volume have been consistently reported in
1129

GLYCOCALYX PERTURBATION IN TYPE 1 DIABETES

FIG. 1. A: Systemic glycocalyx volumes are decreased in type 1 diabetic

patients (DM1-NA and DM1-MA; groups 2 and 3) compared with
matched control subjects (Controls; group 1). Between type 1 diabetic
patients, reduction was highest in DM1-MA compared with DM1-NA.
*P < 0.05 and #P < 0.01. B: Plasma dextran 40 clearance curves in
matched control (), DM1-NA (f), and DM1-MA () subjects. In type
1 diabetes, the rate of dextran 40 plasma clearance is increased
compared with matched control subjects. Values on each time point are
expressed as means SE.

animal models of both acute and chronic hyperglycemia

(26 28).
Possible mechanisms of glycocalyx perturbation. Glycocalyx thickness depends on the rate of synthesis and
shedding of glycosaminoglycans. Over the last years, we
and others (14,29,30) have demonstrated that hyaluronan
is an important component of the endothelial glycocalyx
and that hyperglycemia is associated with increased synthesis of hyaluronan both in humans and in vitro models.
Moreover, recent animal studies (3133) have shown that
the endothelial glycocalyx is an important component of
the glomerular barrier, the magnitude of which firmly
decreases after hyaluronidase infusion. The observation of
both increased hyaluronan and hyaluronidase levels in
both plasma and kidney of diabetic animal models is
consistent with our present finding in type 1 diabetic
subjects, correlating the reduction in glycocalyx volume
with enhanced shedding of these components (29,34). So
far, six hyaluronidase-regulating genes have been identified in humans, of which four are functionally active, and
the presence of renal hyaluronidase acitivity has long been
recognized (35,36). It is tempting to speculate that genetically determined changes in hyaluronan and hyaluroni1130

FIG. 2. A: Capillary glycocalyx thickness is reduced in type 1 diabetes

(DM1-NA and DM1-MA bars) compared with age-matched healthy
control subjects (Controls bar). Individual capillary glycocalyx thickness is estimated from the transient widening of the erythrocyte
column upon compression of the glycocalyx during capillary leukocyte
passage. #P < 0.01. B: Relation between systemic glycocalyx volume
and sublingual capillary glycocalyx thickness in matched control (),
DM1-NA (f), and DM1-MA () subjects.

dase metabolism may contribute to the sensitivity toward

glycocalyx perturbation, which can be associated with the
likelihood of developing vascular dysfunction in type 1
diabetes.
Study limitations. Due to the relatively small sample
size, we were unable to perform multivariate analysis in
order to identify determinants predictive for glycocalyx
damage. In fact, we chose to include a homogenous group
of type 1 diabetic patients with or without microalbuminuria. The clear difference between patient categories and
control subjects underscores potential clinical relevance
of the observation. Secondly, the accuracy of glycocalyx
volume estimates is largely determined by the accuracy of
dextran 40 distribution volume estimates. Because of its
small size and neutral charge, dextran 40 is also cleared
from circulation. Therefore, we estimated the intravascular dextran 40 concentrations before vascular leakage or
renal clearance by extrapolating dextran 40 concentrations to the time of injection. As can be appreciated from
the clearance curve in Fig. 1B, the error of the estimated
initial dextran 40 concentration is relatively small and will
DIABETES, VOL. 55, APRIL 2006

M. NIEUWDORP AND ASSOCIATES

J.A.E.S., M.D., R.J.H., J.B.L.H., J.J.P.K., E.S.G.S., and H.V.

designed the study. M.N., J.K., H.L.M., F.H., and B.A. performed the research. C.I. contributed the analytic tools
(OPS). M.N., E.S.G.S. and H.V. analyzed the data and drafted
the article. All authors critically reviewed the manuscript.
None of the authors have conflicting financial interests.
REFERENCES

FIG. 3. A: Circulating hyaluronan levels increase in a stepwise manner

from control to DM1-NA and DM1-MA subjects. Data are presented as
means SE. *P < 0.05 and #P < 0.01. B: Plasma hyaluronidase activity
is increased in type 1 diabetic patients compared with control subjects.
Data are presented as means SE. *P < 0.05.

therefore have no major impact on the estimates of

glycocalyx volume. Finally, whereas the microvasculature
with its large endothelial surface area contains the majority of systemic glycocalyx volume, the macrovasculature
determines the circulating blood volume. So, decreased
glycocalyx volume with stable blood volume could also
indicate selective loss of microvascular capillary volume.
However, in the present study this scenario is highly
unlikely. Thus, capillary density and dimension between
diabetes without versus diabetes with microalbuminuria
during OPS imaging were not significantly different, in
spite of a significant decline in glycocalyx volume.
Clinical implications. The finding of a gradual reduction
in glycocalyx volume in association with the presence of
microalbuminuria in type 1 diabetic subjects emphasizes
the generalized nature of glycocalyx perturbation in the
development of diabetes-related microvascular disease.
Further studies are needed to address whether glycocalyx
perturbation indicates a poor vascular outcome and
whether restoration of the glycocalyx is a valuable target
to prevent vascular disease progression.
ACKNOWLEDGMENTS

H.V. is an established investigator of the Netherlands

Heart Foundation (dr E. Dekker program 2005 T 037).
J.J.P.K. is a clinical established investigator of the Netherlands Heart Foundation (2000D039).
DIABETES, VOL. 55, APRIL 2006

1. Diabetes Control and Complications Trial Research Group: The effect of

intensive treatment of diabetes on the development and progression of
long-term complications in insulin-dependent diabetes mellitus. N Engl
J Med 329:977986, 1993
2. Nathan DM, Cleary PA, Backlund JY, Genuth SM, Lachin JM, Orchard TJ,
Raskin P, Zinman B, the Diabetes Control and Complications Trial/
Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC)
Study Research Group: Intensive diabetes treatment and cardiovascular
disease in patients with type 1 diabetes. N Engl J Med 353:26432653, 2005
3. Rossing P, Hougaard P, Borch-Johnsen K, Parving HH: Predictors of
mortality in insulin dependent diabetes: 10 year observational follow up
study. BMJ 313:779 784, 1996
4. Kornerup K, Nordestgaard BG, Feldt-Rasmussen B, Borch-Johnsen K,
Jensen KS, Jensen JS: Increased transvascular low density lipoprotein
transport in insulin dependent diabetes: a mechanistic model for development of atherosclerosis. Atherosclerosis 170:163168, 2003
5. Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813 820, 2001
6. Writing Team for the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group:
Sustained effect of intensive treatment of type 1 diabetes mellitus on
development and progression of diabetic nephropathy: the Epidemiology
of Diabetes Interventions and Complications (EDIC) study. JAMA 290:
2159 2167, 2003
7. Hovind P, Tarnow L, Rossing K, Rossing P, Eising S, Larsen N, Binder C,
Parving HH: Decreasing incidence of severe diabetic microangiopathy in
type 1 diabetes. Diabetes Care 26:1258 1264, 2003
8. Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, Jensen T, KofoedEnevoldsen A: Albuminuria reflects widespread vascular damage: the
Steno Hypothesis. Diabetologia 32:219 226, 1989
9. van Haaren PM, VanBavel E, Vink H, Spaan JA: Localization of the
permeability barrier to solutes in isolated arteries by confocal microscopy.
Am J Physiol Heart Circ Physiol 285:H2848 H2856, 2003
10. van den Berg BM, Vink H, Spaan JA: The endothelial glycocalyx protects
against myocardial edema. Circ Res 92:592594, 2003
11. Henry CB, Duling BR: Permeation of the luminal capillary glycocalyx is
determined by hyaluronan. Am J Physiol 277:H508 H514, 1999
12. Nieuwdorp M, Meuwese MC, Vink H, Hoekstra JB, Kastelein JJ, Stroes ES:
The endothelial glycocalyx: a potential barrier between health and vascular disease. Curr Opin Lipidol 16:507511, 2005
13. Constantinescu AA, Vink H, Spaan JA: Endothelial cell glycocalyx modulates immobilization of leucocytes at the endothelial surface. Art Thromb
Vasc Biol 23:15411547, 2003
14. Nieuwdorp M, van Haeften TW, Gouverneur MCLG, Mooij HL, van Lieshout MHP, Levi M, Meijers JCM, Holleman F, Hoekstra JBL, Vink H,
Kastelein JJP, Stroes ESG: Loss of endothelial glycocalyx during acute
hyperglycemia coincides with endothelial dysfunction and coagulation
activation in vivo. Diabetes 55:480 486, 2006
15. Frost GI, Stern R: A microtiter-based assay for hyaluronidase activity not
requiring specialized reagents. Anal Biochem 251:263269, 1997
16. Vink H, Duling BR: Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries.
Circ Res 79:581589, 1996
17. Vink H, Duling BR: Capillary endothelial surface layer selectively reduces
plasma solute distribution volume. Am J Physiol Heart Circ Physiol
278:H285H289, 2000
18. Orth VH, Rehm M, Thiel M, Kreimeier U, Haller M, Brechtelsbauer H,
Finsterer U: First clinical implications of perioperative red cell volume
measurement with a nonradioactive marker (sodium fluorescein). Anesth
Analg 87:1234 1238, 1998
19. 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 275:71 80, 1998
20. Groner W, Winkelman JW, Harris AG, Ince C, Bouma GJ, Messmer K,
Nadeau RG: Orthogonal polarization spectral imaging: a new method for
study of the microcirculation. Nat Med 5:1209 1212, 1999
21. Mathura KR, Vollebregt KC, Boer K, De Graaff JC, Ubbink DT, Ince C:
1131

GLYCOCALYX PERTURBATION IN TYPE 1 DIABETES

Comparison of OPS imaging and conventional capillary microscopy to

study the human microcirculation. J Appl Physiol 91:74 78, 2001
22. Han Y, Weinbaum S, Vink H: Large deformation analysis of the elastic
recoil of fiber layers in a Brinkman medium with application to the
endothelial glycocalyx. J Fluid Mech. In press
23. Klitzman B, Duling BR: Microvascular hematocrit and red cell flow in
resting and contracting striated muscle. Am J Physiol 237:H481H490,
1979
24. Desjardins C, Duling BR: Microvessel hematocrit: measurement and implications for capillary oxygen transport. Am J Physiol 252:H494 H503, 1987
25. Aird WC: Endothelium as an organ system. Crit Care Med 32:S271S279,
2004
26. SextonW, Poole DC, Mathieu-Costello O: Microcirculatory structure-function relationships in skeletal muscle of diabetic rats. Am J Physiol
266:H1502H1511, 1994
27. Kindig CA, Sexton WL, Fedde MR, Poole DC: Skeletal muscle microcirculatory structure and hemodynamics in diabetes. Respir Physiol 111:163
175, 1998
28. Zuurbier CJ, Demirci C, Koeman A, Vink H, Ince C: Short-term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases
lineal density of of capillaries with flowing RBCs. J Appl Physiol 99:1471
1476, 2005

1132

29. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N, Takahashi T: Enhanced

activity of serum and urinary hyaluronidases in streptozotocin-induced
diabetic Wistar and GK rats. Glycobiology 14:6572, 2004
30. Wang A, Hascall VC: Hyaluronan structures synthesized by rat mesangial
cells in response to hyperglycemia induce monocyte adhesion. J Biol
Chem 279:10279 10285, 2004
31. Deen WM: What determines glomerular capillary permeability? J Clin
Invest 114:14121414, 2004
32. Jeansson M, Haraldsson B: Glomerular size and charge selectivity in the
mouse after exposure to glucosaminoglycan-degrading enzymes. J Am Soc
Nephrol 14:1756 1765, 2003
33. Jeansson M, Haraldsson B: Morphological and functional evidence for an
important role of the endothelial cell glycocalyx in the glomerular barrier.
Am J Physiol Renal Physiol 290:F111F116, 2006
34. Ikegami-Kawai M, Suzuki A, Karita I, Takahashi T: Increased hyaluronidase
activity in the kidney of streptozotocin-induced diabetic rats. J Biochem
134:875 880, 2003
35. Csoka AB, Frost GI, Stern R: The six hyaluronidase-like genes in the
human and mouse genomes. Matrix Biol 20:499 508, 2001
36. Bollet AJ, Bonner WM, Nance JL: The presence of hyaluronidase in various
mammalian tissues. J Biol Chem 238:35223527, 1963

DIABETES, VOL. 55, APRIL 2006