UvA-DARE (Digital Academic Repository)
Crossing borders: the role of the endothelial glycocalyx and intravascular
haemostasis in vascular complications of diabetes mellitus
Lemkes, B.A.
Publication date
2011
Link to publication
Citation for published version (APA):
Lemkes, B. A. (2011). Crossing borders: the role of the endothelial glycocalyx and
intravascular haemostasis in vascular complications of diabetes mellitus.
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CHAPTER
4
Mild hyperglycaemia disturbs vascular homeostasis in humans
Bregtje A. Lemkes, Sarah E. Siegelaar, Joost C.M. Meijers, Wim Kulik, Hans Vink, Joost B.L. Hoekstra, Max Nieuwdorp and Frits Holleman
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Abstract
Hyperglycaemia induces oxidative stress, disturbs endothelial function, damages the endothelial glycocalyx and causes a prothrombotic shift in coagulation and fibrinolysis. Little is known about the exact blood glucose level necessary to start these processes. The aim of this study was to determine at which level of glycaemia these changes occur. A stepwise hyperglycaemic clamp was performed in eleven healthy human males at blood glucose (BG) levels of 6, 8 and 10 mmol/l for two hours each while suppressing endogenous insulin release. Oxidative stress, assessed by malondialdehyde, showed a gradual increase highly correlating with BG. Coagulation, assessed by prothrombin fragment F1+2, significantly increased at 6 mmol/l and was followed by an increase in both plasmin-antiplasmin complexes and d-dimer levels at 8 mmol/l, indicating fibrinolysis activation. Imaging of the glycocalyx and hyaluronic acid levels showed no relevant changes during the clamp. Hyaluronidase showed a gradual decrease indicating increased hyaluronidase substrate binding by shedding of glycocalyx constituents, significant at 10 mmol/l. These data indicate that oxidative stress and coagulation activation already start at near normal BG levels, while endothelial glycocalyx changes occur at 10 mmol/l.
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fects of mild hyper
glycaemia
on vascular homeostasis
Patients with diabetes mellitus are at high risk of developing cardiovascular disease. A combination of accelerated atherosclerosis and a shift towards a pro-coagulant state
leads to atherothrombotic events in nearly two thirds of all patients with diabetes1.
Hyperglycaemia, its defining feature, has been shown to cause both endothelial
dysfunction, a precursor of atherosclerosis, and activation of the coagulation system2,
3. Endothelial dysfunction is paired with damage to the endothelial glycocalyx, the
protective layer of proteoglycans and glycosaminoglycans lining the luminal side of all blood vessels. Hyperglycaemia induced disruption of the endothelial glycocalyx results in a pro-atherogenic state, characterized by increased vascular permeability, coagulation
activation and increased cellular adhesion and migration4. The formation of reactive
oxygen species (ROS) is an important mechanism by which hyperglycaemia leads to endothelial (glycocalyx) damage, since high doses of anti-oxidants are able to attenuate
this damage5. Furthermore, hyperglycaemia induced ROS formation may also affect the
coagulation system, by influencing gene transcription of coagulation and fibrinolytic
factors6, 7.
It is unclear, however, at which glucose level these vascular changes first occur and whether this is an on-off phenomenon with a threshold or a continuous relationship. This distinction is also of importance given the recent debate about the impact of glycaemic
variability on the development of complications in patients with diabetes8, 9. Some have
argued that a high variation in blood glucose levels throughout the day has a greater
impact on pro-atherogenic processes than a stable high glucose10-12. If so, a threshold
phenomenon should exist for these processes, since a dose-dependent effect would lead to comparable outcome when the mean blood glucose levels are equal.
Most studies investigating the effects of hyperglycaemia on vascular homeostasis have
described effects of a glucose level of 10 mmol/l or higher2, 5, but epidemiological studies
suggest that vascular damage actually starts at near normal glucose levels. Even in the lower glucose ranges a linear relationship between HbA1c, fasting plasma glucose and vascular complications of diabetes was demonstrated in patients with both type 1 and
type 2 diabetes13,14. Moreover, impaired glucose tolerance and impaired fasting glucose,
both representing only mildly elevated glucose levels, already carry an increased risk for
macrovascular disease15.
In the present study we describe the effects of only mildly elevated glucose levels on oxidative stress, the endothelial glycocalyx and the thrombotic system in healthy males, studied by performing a stepwise glucose clamp while suppressing endogenous insulin levels.
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Results
ProtocolWe investigated whether near-normal glucose levels were associated with endothelial dysfunction by means of a stepwise hyperglycaemic clamp while suppressing endogenous insulin production by octreotide infusion. Blood glucose (BG) levels were maintained at 6, 8 and 10 mmol/l successively for 2 hours per level (Figure 1). We obtained blood samples and performed sidestream dark-field (SDF) imaging to assess endothelial glycocalyx dimensions every 30 minutes during the clamp. Also, the day after the clamp a fasting blood sample was obtained and SDF imaging was performed to assess the recovery after the glucose load.
Figure 1. Glucose clamp
Glucose values obtained during the clamp. Data are expressed as mean of the previous 30 minutes with its standard deviation. Dotted lines represent the time points where glucose infusion was increased to go to the next glucose level.
Patients
In total, 14 healthy non-smoking Caucasian males with a fasting plasma glucose level ≤5 mmol/l without risk factors for macrovascular disease as measured by BMI, blood pressure, cholesterol and triglyceride levels were included in the study. Of those, one dropped out due to febrile illness before the study day and two subjects were excluded before analysis of the blood samples due to poor performance of the hyperglycaemic clamp, resulting in 11 subjects who were included in the final analyses. Baseline characteristics of the included subjects are listed in Table 1.
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Table 1. Baseline characteristics
n=11
Age, years 24.3 (3.6)
BMI, kg/m2 21.6 (1.9)
Systolic blood pressure, mmHg 115.1 (12.3) Diastolic blood pressure, mmHg 68.9 (8.5) Fasting plasma glucose, mmol/l 4.7 (0.2)
HbA1c, % 5.3 (0.2)
Total cholesterol, mmol/l 4.0 (0.9) LDL cholesterol, mmol/l 2.2 (0.9) HDL cholesterol, mmol/l 1.5 (0.3) Triglycerides, mmol/l 0.7 (0.4)
Values are expressed as means (SD). BMI, body mass index; HDL, high density lipoprotein; LDL, low density lipoprotein
Hyperglycaemic clamp
During the clamp endogenous insulin levels were adequately suppressed. Plasma insulin levels after 1-hr of octreotide infusion and at the end of the clamp were comparable with fasting levels in all patients (median <15 pmol/l, maximum 43 pmol/l). Mean glucose levels of all included time points are depicted in Figure 1.
Oxidative stress
Oxidative stress was assessed by quantitative determination of malondialdehyde (MDA) in plasma using HPLC tandem mass spectrometry. MDA is a reactive and potentially mutagenic aldehyde which is formed as a result of lipid peroxidation caused by hyperglycaemia induced formation of ROS. Lipid peroxidation is thought to be an
important part of the pathogenesis of atherosclerosis16 and the metabolites are frequently
used as biomarkers for oxidative stress17.
Plasma MDA levels during the glucose clamp are depicted in Figure 2 and Table 2. Plasma MDA levels did not increase after 1-hr octreotide infusion (median [IQR] 6.6 µmol/l [6.2-7.7] to 6.8 µmol/l [6.0-8.0], p=0.89). After the start of the glucose infusion, plasma MDA increased gradually accompanying the increase in blood glucose with a strong correlation between MDA and blood glucose levels (ρ=0.82, p<0.001, Spearman correlation). Median MDA levels at the 8 mmol/l glucose plateau were significantly higher than after 1-hr octreotide infusion (9.9 µmol/l [9.3-10.6], p=0.02) and were further increased substantially at the 10 mmol/l plateau (11.8 µmol/l [10.8-12.5], p=0.01). No cumulative effect of glucose over time during each plateau was observed (Friedman test for repeated measures). The day after the clamp median plasma MDA levels had returned to baseline (6.4 µmol/l [5.6-6.9], p=0.24).
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Plasma levels of the parameters of inter
est during the glucose clamp
Marker Unit Baseline T=1h 6 mmol/l 8 mmol/l 10 mmol/l T=24h MDA µmol/l 6.6 (6.2-7.7) 6.8 (6.0-8.0) 8.0 (7.6-9.6) 9.9 (9.3-10.6)* 11.8 (10.8-12.5)* 6.4 (5.6-6.9) F1+2 pmol/l 140 (1 18-151) 170 (124-474) 508 (275-1714)* 731 (446-1095)* 608 (445-817)* 136 (123-169) vWF % 66 (50-127) 63 (53-1 13) 59 (39-86)* 58 (39-107)* 55 (42-78)* 76 (58-130) ETP nM.min 1271 (1 167-1415) 1237 (1074-1429) 1297 (1082-1425) 1263 (1 137-1440) 1296 (1 143-1486)* 1328 (1 170-1481)^ Peak thr ombin nM 222 (200-264) 209 (186-244) 208 (180-245) 187 (174-245) 209 (189-243) 226 (21 1-265) PA P mg/l 366 (268-460) 335 (244-934) 540 (271-854) 603 (398-1060)* 615 (508-71 1) 426 (277-650) d-dimer mg/l 0.00 (0-0.18) 0.00 (0-0.05) 0.08 (0-0.23) 0.28 (0-0.51)* 0.36 (0.04-0.51)* 0.19 (0-0.39)^ HA ng/ml 49.6 (48.1-50.2) 49.5 (47.9-50.2) 49.9 (48.8-50.9) 49.9 (47.0-51.5)* 50.3 (47.2-51.2)* 51.6 (50.4-54.5)^ Hyalur onidase U/ml 51.6 (43.6-55.3) 49.4 (44.0-58.3) 45.4 (36.3-48.6) 48.3 (35.4-55.2) 36.0 (33.4-41.4)* 49.5 (44.1-53.0) At each glucose plateau the median (IQR) values of the mean value per patient ar e depicted. MDA, malondialdehyde; F1+2, pr othr ombin fragment 1+2; vWF , von W illebrand factor; P AP
, plasmin-antiplasmin complex; ETP
, endogenous thr
ombin potential; HA, hyalur
onic acid.
* p<0.05 compar
ed to T=1
^ p<0.05 compar
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fects of mild hyper
glycaemia
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Figure 2. Oxidative stress
Oxidative stress was assessed by plasma malondialdehyde (MDA) levels during the glucose clamp. Data are depicted as medians with interquartile ranges. Dotted lines represent the time points where glucose infusion was increased to go to the next glucose level. *p<0.05 compared to T=1, after 1-hr of octreotide infusion.
Coagulation
We determined the effects of increasing glucose levels on coagulation by measuring prothrombin fragment 1+2 (F1+2) and von Willebrand factor (vWF). F1+2 are released when thrombin is formed from prothrombin and therefore provide an in vivo measure of thrombin formation. VWF plays a major role in haemostasis by ensuring the arrest of blood platelets at sites of injury, and by binding of coagulation factor VIII, but it is also an established marker of endothelial dysfunction. The effects of the stepwise increase in blood glucose on these markers of coagulation are depicted in Figure 3 and Table 2. No significant effect on F1+2 levels or vWF was detected after octreotide infusion. Median F1+2 levels showed a significant increase from 170 pmol/l (124-474) to 508 pmol/l (275-1714) when the glucose level was raised to 6 mmol/l, further increased to 731 pmol/l (446-1095) at 8 mmol/l and remained at a stable high level at 10 mmol/l with no significant differences between the glucose plateaus. The following day F1+2 levels had returned to baseline. After raising the glucose level to 6 mmol/l vWF levels dropped to 59% (39-86, p=0.02). An increase of glucose to 8 mmol/l led to a further decrease of vWF levels to 58% (39-107, p=0.05), but raising the glucose level to 10 mmol/l did not cause further significant changes. After 24 hours, vWF levels had returned to baseline values. Finally, we determined the endogenous thrombin potential (ETP), which represents the balance between pro- and anti-coagulant processes in plasma and provides an ex vivo measure for overall coagulability. No significant effects of the octreotide run-in period or any of the blood glucose levels on peak thrombin values could be detected (Table 2). ETP showed a significant decrease from 1271 nM.min (1167-1415) to 1237 nM.min (1074-1429) after the 1-hour octreotide period (p=0.01) and a subsequent increase to 1296 nM.min
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(1143-1486) at the 10 mmol/l glucose plateau (p=0.01). The following day, ETP remained higher than baseline at 1328 nM.min (1170-1481, p=0.05).
Figure 3. Coagulation
Coagulation was assessed by prothrombin fragment 1+2 (F1+2; left panel) and von Willebrand factor (vWfag; right panel) plasma levels. Data are depicted as medians with interquartile ranges. Dotted lines represent the time points where glucose infusion was increased to go to the next glucose level. *p<0.05 compared to T=1, after 1-hr of octreotide infusion.
Fibrinolysis
Fibrinolysis was assessed by measuring plasmin-alpha2-antiplasmin (PAP) complexes and d-dimer (Figure 4 and Table 2). PAP complexes serve as an indicator of recent in vivo fibrinolytic activity, since alpha2 antiplasmin is the most important circulating inhibitor of plasmin, the main enzyme in the fibrinolytic system. D-dimer is a fibrin degradation product, which is dependent on the amount of fibrin that is generated (coagulation) as well as the ability of the fibrinolytic system to degrade the generated fibrin (fibrinolysis). Neither PAP complexes nor d-dimer had changed significantly after the 1-hr octreotide infusion period (T=1; figure 4). Median PAP levels were not significantly different at a blood glucose level of 6 mmol/l when compared to T=1, but did show a significant increase at a blood glucose of 8 mmol/l (335 µg/l [244-934] to 603 µg/l [398-1060], p=0.01). PAP levels remained at a stable high level when blood glucose was further increased to 10 mmol/l and returned to baseline the following day. Median D-dimer levels showed an increasing trend from 0.00 mg/l (0.00-0.05) to 0.08 mg/l (0.00-0.23) when the glucose level was raised to 6 mmol/l (p=0.07). At 8 mmol/l d-dimer levels had risen to 0.28 mg/l (0.00-0.51; p=0.04, compared to T=1) and increased further to 0.36 mg/l (0.004-0.51) when blood glucose was raised to 10 mmol/l (p=0.02, compared to T=1). After 24 hours, d-dimer levels remained higher than at T=0, at 0.19 mg/l (0.00-0.39; p=0.04).
No cumulative effect of any of the coagulation and fibrinolysis parameters was detected at the three examined blood glucose levels, except for F1+2 at a blood glucose level of 6
mmol/l (p=0.02, Friedman test for repeated measures).
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fects of mild hyper
glycaemia
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Figure 4. Fibrinolysis
Fibrinolysis was assessed by plasmin-alpha-antiplasmin (PAP) complexes (left panel) and d-dimer (right panel) plasma levels. Data are depicted as medians with interquartile ranges. Dotted lines represent the time points where glucose infusion was increased to go to the next glucose level. *p<0.05 compared to T=1, after 1-hr of octreotide infusion.
Endothelial glycocalyx
The dimension of the endothelial glycocalyx in individual capillary blood vessels (< 50 µm in diameter) was assessed by sidestream dark-field (SDF) imaging of the sublingual microcirculation, which visualizes flowing erythrocytes in the sublingual capillary network. Perturbation of the endothelial glycocalyx results in an impairment of its
red blood cell excluding properties18, 19, allowing red blood cells to approach the vessel
wall. This can be measured as change in median red blood cell column width in the microcirculation by SDF imaging (Figure 5). Additionally, we assessed the outer edge of the red blood cell perfused lumen at each measurement site, the RBC perfused diameter, by linear extrapolation of all RBC column width percentiles between P25 and P75 (Figure 6A). This allowed for the determination of the Perfused Boundary Region, which includes the cell permeable part of the glycocalyx, by calculating the distance of the median RBC column width to the outer edge of the extrapolated perfused diameter. Throughout the experiment no significant changes in median RBC width, perfused diameter or perfused boundary region were detected by SDF imaging (Figure 6B).
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Figure 5. Endothelial glycocalyx imaging method
Imaging method of the endothelial glycocalyx. Left panel: Perturbation of the endothelial glycocalyx allowing RBC’s to approach the vessel wall, or invade the perfused boundary region, thereby changing RBC width. Right panel: Distribution of all RBC column width measurements, e.g. at P25 25% of all RBC column width measurements was 5 µm or less and 75% was larger than 5 µm. Linear extrapolation of all RBC column width percentiles between P25 and P75 to obtain the perfused boundary region. CFL, cell free region; Gx, endothelial glycocalyx; PBR, perfused boundary region; RBC, red blood cell.
Figure 6. Endothelial glycocalyx dimension
Endothelial glycocalyx dimension was assessed by the median RBC width, the perfused diameter and as a result the perfused boundary region (see Figure 5 for explanation of the imaging method). Data are depicted as means with standard deviation. Dotted lines represent the time points where glucose infusion was increased to go to the next glucose level. *p<0.05 compared to T=1, after 1-hr of octreotide infusion. RBC, red blood cell.
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57
The effect of elevation of blood glucose levels on the endothelial glycocalyx was also assessed by plasma measurement of its main component hyaluronic acid (HA) and its regulatory enzyme hyaluronidase, to detect shedding from the glycocalyx. Both were unaffected by 1-hr octreotide infusion (T=1). When raising the blood glucose level to 6 mmol/l, median HA levels remained unaffected but a raise to 8 and 10 mmol/l showed a significant, but small increase compared to T=1 (from 49.5 ng/ml [47.9-50.2] to 49.9 ng/ ml [47.0-51.5], p=0.038, and to 50.3 ng/ml [47.2-51.2], p=0.008). This increase persisted after 24 hours. Plasma hyaluronidase activity showed a gradual decrease during the clamp, with significantly lower activity at a blood glucose level of 10 mmol/l (36.0 [33.4-41.4] U/ml compared to 51.6 [43.6-55.3] U/ml at T=1, p=0.005). After 24-hours, these levels had returned to baseline values (Figure 7 and Table 2).
Figure 7. Endothelial glycocalyx shedding
Shedding of endothelial glycocalyx components was assessed by plasma hyaluronan levels (left panel) and activity of the regulatory enzyme hyaluronidase (right panel). Data are depicted as medians with interquartile ranges. Dotted lines represent the time points where glucose infusion was increased to go to the next glucose level. *p<0.05 compared to T=1, after 1-hr of octreotide infusion.
Discussion
In this study we show that oxidative stress, represented by MDA levels, showed a stepwise increase during the clamp, mimicking the glucose curve. The coagulation system was activated even at near normal glucose levels of 6 mmol/l, resulting in a significant increase in prothrombin fragment 1+2 (F1+2) indicating thrombin formation. This was followed by activation of the fibrinolytic system, as measured by PAP complexes and d-dimer, at a glucose level of 8 mmol/l. Relevant endothelial glycocalyx dimensional changes were not detected using both imaging and biochemistry techniques, except for a decrease in hyaluronidase activity when the glucose concentration was raised to 10 mmol/l.
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To our knowledge this is the first study examining the effects of isolated mild hyperglycaemia, with a maximum of 10 mmol/l, on vascular homeostasis. Previous studies on oxidative stress show glucose dependent ROS formation with blood glucose
levels above 10 mmol/l2, 10 which is comparable with our findings and in vitro studies20.
Our data suggest that hyperglycaemia dependent ROS formation is dose-dependent rather than an on-off phenomenon. This is depicted in Figure 2 showing no cumulative effect within the different glucose plateaus but only an increase in oxidative stress when blood glucose is increased to the next level. Data reported by Ceriello and colleagues support this finding showing higher plasma nitrotyrosine levels at a plasma glucose of 15 mmol/l compared to 11 mmol/l as well as no further increase in plasma nitrotyrosine
levels when stabilizing plasma glucose10. Also, in type 2 diabetes patients an impressive
correlation between MDA and blood glucose, ranging from 6 to 14 mmol/l, was found
after a mixed-meal test suggesting an insulin-independent effect21.
Our results are in line with previous observations, which have shown that hyperglycaemia
activates the coagulation5, 22 as well as the fibrinolytic system5. Unlike oxidative stress,
our results indicate that the glucose induced-activation of the coagulation system is an on-off phenomenon showing a more than threefold increase in thrombin generation, measured by F1+2 levels, triggered by a blood glucose level of only 6 mmol/l. This hypothesis is supported by the observation that maximum levels are reached quickly and show no increase, perhaps even a decrease, at the highest glucose level. Moreover, the maximum levels of F1+2 and d-dimer are comparable with the levels reached during a hyperglycaemic clamp at a blood glucose of 15 mmol/l previously performed by our
group5. The timing of the increase in fibrinolytic activity, closely following the coagulant
activity, suggests that the increased fibrinolytic activity is secondary to the coagulation
activation. Conversely, diabetes mellitus is associated with impairment in fibrinolysis23,
which we did not detect in our study. However, Stegenga and colleagues showed that
fibrinolysis was mainly affected by hyperinsulinaemia as opposed to hyperglycemia22,
and insulin was suppressed throughout our experiments. ETP changed only minimally during and after the clamp. This indicates no relevant change in the thrombin generating capacity of the coagulation system itself, but rather suggests that glucose is a trigger for the in vivo activation of coagulation. VWF levels showed a maximal decrease of 5%. This modest change could be due to increased binding to blood platelets, known to be activated by hyperglycaemia, or caused by physical inactivity of the participants.
VWF levels have been shown to increase after physical exercise24 and previous control
experiments by our group have shown a similar decreasing effect of a 6-hour saline infusion in healthy males (M. Nieuwdorp, unpublished data).
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We did not detect changes in glycocalyx dimension or plasma HA levels and a decrease in hyaluronidase activity was found only at a glucose level of 10 mmol/l. Previous investigations show marked increase in HA shedding from 70 ng/ml at baseline to 112
ng/ml with blood glucose levels of 15 mmol/l5, suggesting that the trigger for direct
endothelial damage as reflected by loss of glycocalyx lies above a blood glucose level of 10 mmol/l. Statistically, there was a change in plasma HA levels at 8 and 10 mmol/l. However, the maximum increase was only 1.6% indicating no significant biological effect. This is supported by the limited effects on vWF levels, which are considered a marker for endothelial damage. The decrease in hyaluronidase activity at 10 mmol/l does indicate substrate binding to this enzyme. This substrate may consist of other glycosaminoglycans than HA shed from the glycocalyx, such as heparan sulphate or chondroitin sulphate, since these are also bound by hyaluronidase. The contribution of these glycosaminoglycans to the dimension of the endothelial glycocalyx may be too small to detect dimensional changes by SDF imaging at these glucose levels.
The results of our study are in line with epidemiological data, which show that the
increase in cardiovascular risk already starts at mildly elevated glucose levels13-15.
Nonetheless, our results indicate that glucose-induced activation of the coagulation system and ROS formation are completely reversible after 24 hours. Therefore, these changes may not be considered to be pathological in healthy subjects who spent the
greater part of the day with glucose levels below 6.1 mmol/l25. Conversely, patients with
diabetes or pre-diabetes by definition have a fasting glucose level of > 5.6 mmol/l26 if
untreated, and are exposed to glucose levels above 6 mmol/l throughout the day. This may interfere with the reversibility of the changes in coagulation and oxidative stress, and translate to pathological effects. Moreover, in diabetes inappropriate activation of the coagulation system may not be counterbalanced because of the fibrinolytic impairment
associated with this disease23. Our results do not support a role for glucose variability in
coagulation activation and ROS formation, since coagulation activation occurred even at a blood glucose of 6 mmol/l and the relationship of glucose levels with oxidative stress was continuous.
Several aspects of our study need comment. First, this study was specifically designed to assess the effects of mild hyperglycaemia on several components of vascular homeostasis and was therefore performed under full suppression of insulin levels. In disease states, such as type 2 diabetes or stress-hyperglycaemia during severe illness, high glucose levels are accompanied by high insulin levels and therefore our results cannot be extrapolated directly to these settings. However, glucose levels are highly predictive of vascular
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Second, given the design of our experiment, it can be argued that the effects we detected may not be glucose specific, but rather result from the osmotic effect of raising blood glucose or from prolonged administration of octreotide. However, previous work from our group has shown no effect on coagulation or fibrinolysis of a control experiment during which octreotide was administered in combination with mannitol infusion for six hours, serving as a time and osmolality control. Moreover, in our study no significant effect on any of the parameters after one hour of octreotide infusion was detected. This is supported by literature, showing no significant vaso-active or haemostatic effects of this
dose of octreotide28, 29.
In conclusion, our results show that glucose-induced changes to vascular homeostasis already start at near normal glucose levels. Furthermore our study reveals a dose-dependent effect of glucose on MDA formation and an on-off phenomenon for glucose induced coagulation activation, while changes to the endothelial glycocalyx occur at glucose levels of 10 mmol/l or higher. These results give us more insight in the glucose driven mechanisms of vascular complications in humans. To elucidate the difference between acute and chronic mild hyperglycaemia on vascular homeostasis, further studies are needed.
Methods
Patients
The study was approved by the institutional medical ethical committee and conducted according to Declaration of Helsinki principles. Participants signed informed consent prior to inclusion after oral and written explanation of the study.
Stepwise hyperglycaemic clamp protocol (Figure 8)
After an overnight fast, two catheters for venous access were placed, one in every arm. First, basal measurements of glycocalyx dimension, haemostasis and ROS formation were performed. Octreotide was dissolved in saline 0.9% and albumin 20% (proportion 59:1 in a 60 ml syringe) and administered at a final concentration of 30 ng/kg/min
octreotide, to suppress endogenous insulin production5. To confirm that this infusion
did not influence the parameters of interest, the basal measurements were repeated after 60 minutes of octreotide infusion. Hereafter, glucose infusion with 20% glucose solution was started to reach the desired glucose concentration based on a steady state
principle30. The plasma glucose concentration was held constant at the desired plateau
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glycaemia
on vascular homeostasis
and adjusting the glucose solution accordingly. When a stable glucose concentration was reached, glycocalyx dimension, ROS formation and haemostasis parameters were measured every 30 minutes; 4 times per plateau, the last measurement being the baseline value of the next step. Glucose infusion was then increased to reach the next level of glycaemia and measurements were repeated. In total, the actual clamp took 420 minutes. Blood samples were centrifuged within 1 hour after collection and stored at -80°C. Figure 8. Glucose clamp protocol
Depicted in the lower boxes are the times from baseline where the several actions were performed. An arrow indicates an assessment point for oxidative stress, glycocalyx and coagulation/fibrinolysis parameters.
Oxidative stress
Plasma MDA concentration was determined using high liquid performance
chromatography (HLPC) tandem mass spectrometry as described by Pilz31, with minor
modifications. Total (free and bound) malondialdehyde (MDA) in human plasma samples was determined as the 2,4-dinitrophenylhydrazine (DNPH) derivative. After addition of
the stable isotopically labelled analogue (2H
2-MDA) as internal standard (IS), alkaline
hydrolyzation, deproteinization and derivatization with DNPH, MDA-hydrazone was analyzed by HPLC-MS/MS and positive electrospray ionization. Using an Acquity UPLC system (Waters Corporation, Milford, MA), samples were injected on a LC-18-DB analytical column (250 × 4.6 mm, 5 µm particles, Supelco) hyphenated to a Quattro Premier XE mass spectrometer (Waters Corporation, Milford, MA). Analytes and IS were eluted with acetonitrile/water/acetic acid (50/50/0.2) and detected in multiple reaction monitoring (MRM) mode for the transitions m/z 235 → m/z 159; m/z 237 → m/z 161. Samples were quantified against calibration standards.
Fasting
≤5.0 mmol/l 6.0 mmol/l 8.0 mmol/l 10.0 mmol/l
T=0h Start: Octreotide T=1h Start: Glucose T=7h Stop: Octreotide Glucose T=3h Glucose↑ Glucose↑ T=5h
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Sidestream darkfield imaging (SDF) was performed using a Microscan handheld camera
(MicroVision Medical Inc., Wallingford, PA, USA.) as described previously32.
Video sequences of 2 seconds each were recorded using Streampix software (Norpix Inc. Montreal, Canada) in at least 10 areas close to the frenulum. Next, all vessels with a diameter of <50 µm were automatically selected and measurement lines perpendicular to the vessel direction were placed every 10 µm along each visible microvessel. At each measurement site 21 parallel intensity profiles were plotted using Image J software (National Institutes of Health, Bethesda, MD) for 40 consecutive frames, resulting in 840 RBC column width measurements at each site.
Reproducibility data was acquired by performing SDF imaging on two separate days in 16 male volunteers. Participants did not smoke, did not use any medication, and were free from any illness, including overt cardiovascular disease. All experiments were performed after an overnight fast. Group averages (SD) for RBCW, PBR and PD are RBCW_V01: 10.09 µm (1.23), RBCW_V02: 10.05 µm (1.06); PBR_V01: 2.72 µm (0.59), PBR_V02: 2.59 µm (0.50); PD_V01: 16.01 µm (1.19), PD_V02: 15.67 µm (1.38).
Hyaluronic acid was measured by a commercially available ELISA kit (Corgenix, Inc., Broomfield, Colorado, USA). In short, HA reacted with hyaluronic acid binding protein. Thereafter horseradish peroxidase was added to form complexes with bound HA. After addition of a chromogenic substrate the intensity of the colour was measured in optical density units with a spectrophotometer at 450 nm. Hyaluronidase activity was
determined by a previously described assay19, 33.
Coagulation and fibrinolysis
D-dimer was measured on an automated coagulation analyzer (Behring Coagulation System, BSC) using protocols and reagents from the manufacturer (Siemens Healthcare Diagnostics, Marburg, Germany). Antigen levels of vWF were assayed by ELISA using antibodies from Dako (Glostrup, Denmark). Prothrombin fragment 1+2 and PAP were determined by ELISA from Siemens Healthcare Diagnostics and DRG (Marburg, Germany), respectively. The endogenous thrombin potential was determined using
the Calibrated Automated Thrombogram as described by Hemker et al.34 and the
Thrombinoscope manual (Maastricht, the Netherlands). Coagulation was triggered by recalcification in the presence of 5 pM recombinant human tissue factor (Innovin, Siemens Healthcare Diagnostics), 4 µM phospholipids, and 417 µM fluorogenic substrate Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland). Fluorescence was monitored using the Fluoroskan Ascent fluorometer (ThermoLabsystems, Helsinki, Finland), and the ETP and peak thrombin were calculated using the Thrombinoscope software.
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fects of mild hyper
glycaemia
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Data interpretation
The study was conducted to assess the influence of a certain level of plasma glucose on the parameters described above. We excluded samples taken at a certain glucose plateau when the desired glucose level was exceeded by more than 1 mmol/l since crossing the desired glucose level could interfere with the study results. For example, when a plasma glucose level of 7.1 mmol/l occurred at any point during the 6 mmol/l plateau phase, all subsequent samples taken at the 6 mmol/l plateau were excluded from analysis. Moreover, samples were only included in the analysis when the desired glucose level was truly reached. This was determined by calculation of the mean glucose level of the 30 minutes before sampling which had to be within 0.5 mmol/l of the desired glucose level.
Statistical analysis
Baseline characteristics are expressed as mean (SD) and outcome parameters as median (IQR). Differences between plateaus were assessed by a Wilcoxon signed ranks test for paired data. The influence of time on the measurements at each glucose level was assessed using the Friedman test. All analyses were performed using Predictive Analytics Software (PASW) statistics version 18.0 (SPSS Inc., Chicago, IL, USA). A p-value <0.05 was considered statistically significant.
Acknowledgements
We would like to thank Jeroen Sierts from the laboratory of Experimental Vascular Medicine and Arno van Cruchten from the laboratory Genetic Metabolic Diseases, both at the Academic Medical Centre, Amsterdam, the Netherlands, for their excellent laboratory support.
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