Effect of sulodexide on endothelial glycocalyx and vascular permeability in patients with type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Endothelial glycocalyx perturbation contributes to increased vascular permeability. In the present study we set out to evaluate whether: (1) glycocalyx is perturbed in individuals with type 2 diabetes mellitus, and (2) oral glycocalyx precursor treatment improves glycocalyx properties. METHODS: Male participants with type 2 diabetes (n = 10) and controls (n = 10) were evaluated before and after 2 months of sulodexide administration (200 mg/day). The glycocalyx dimension was estimated in two different vascular beds using sidestream dark field imaging and combined fluorescein/indocyanine green angiography for sublingual and retinal vessels, respectively. Transcapillary escape rate of albumin (TER(alb)) and hyaluronan catabolism were assessed as measures of vascular permeability. RESULTS: Both sublingual dimensions (0.64 [0.57-0.75] µm vs 0.78 [0.71-0.85] µm, p < 0.05, medians [interquartile range]) and retinal glycocalyx dimensions (5.38 [4.88-6.59] µm vs 8.89 [4.74-11.84] µm, p < 0.05) were reduced in the type 2 diabetes group compared with the controls whereas TER(alb) was increased (5.6 ± 2.3% vs 3.7 ± 1.7% in the controls, p < 0.05). In line with these findings, markers of hyaluronan catabolism were increased with diabetes (hyaluronan 137 ± 29 vs 81 ± 8 ng/ml and hyaluronidase 78 ± 4 vs 67 ± 2 U/ml, both p < 0.05). Sulodexide increased both the sublingual and retinal glycocalyx dimensions in participants with diabetes (to 0.93 [0.83-0.99] µm and to 5.88 [5.33-6.26] µm, respectively, p < 0.05). In line, a trend towards TER(alb) normalisation (to 4.0 ± 2.3%) and decreases in plasma hyaluronidase (to 72 ± 2 U/ml, p < 0.05) were observed in the diabetes group. CONCLUSION/INTERPRETATION: Type 2 diabetes is associated with glycocalyx perturbation and increased vascular permeability, which are partially restored following sulodexide administration. Further studies are warranted to determine whether long-term treatment with sulodexide has a beneficial effect on cardiovascular risk. TRIAL REGISTRATION: www.trialregister.nl NTR780/ http://isrctn.org ISRCTN82695186 FUNDING: An unrestricted Novartis Foundation for Cardiovascular Excellence grant (2006) to M. Nieuwdorp/E. S. G. Stroes, Dutch Heart Foundation (grant number 2005T037).

In vivo assessment of the human cerebral microcirculation and its glycocalyx: A technical report.

INTRODUCTION: The cerebral microcirculation and its glycocalyx, a matrix coating the luminal endothelium, are key regulators of capillary permeability and cerebral blood flow. Microvascular abnormalities are described in several neurological disorders. However, assessment of the cerebral microcirculation and glycocalyx has mainly been performed ex vivo. NEW METHOD: Here, the technical feasibility of in vivo assessment of the human cerebral microcirculation and its glycocalyx using sidestream dark field (SDF) imaging is discussed. Intraoperative assessment requires the application of a sterile drape covering the camera (slipcover). First, sublingual measurements with and without slipcover were performed in a healthy control to assess the impact of this slipcover. Subsequently, using SDF imaging, the sublingual (reference), cortical, and hippocampal microcirculation and glycocalyx were evaluated in patients who underwent resective brain surgery as treatment for drug-resistant temporal lobe epilepsy. Finally, vessel density, and the perfused boundary region (PBR), a validated gauge of glycocalyx health, were calculated using GlycoCheck© software. RESULTS: The addition of a slipcover affects vessel density and PBR values in a control subject. The cerebral measurements in five patients were more difficult to obtain than the sublingual ones. This was probably at least partly due to the introduction of a sterile slipcover. Results on vessel density and PBR showed similar patterns at all three measurement sites. COMPARISON WITH EXISTING METHODS: This is the first report on in vivo assessment of the human cerebrovascular glycocalyx. Assessment of the glycocalyx is an additional application of in vivo imaging of the cerebral microcirculation using SDF technique. This method enables functional analysis of the microcirculation and glycocalyx, however the addition of a sterile slipcover affects the measurements. CONCLUSIONS: SDF imaging is a safe, quick, and straightforward technique to evaluate the functional cerebral microcirculation and glycocalyx. Because of their eminent role in cerebral homeostasis, this method may significantly add to research on the role of vascular pathophysiology underling various neurological disorders.

Protocol for intraoperative assessment of the human cerebrovascular glycocalyx.

INTRODUCTION: Adequate functioning of the blood-brain barrier (BBB) is important for brain homoeostasis and normal neuronal function. Disruption of the BBB has been described in several neurological diseases. Recent reports suggest that an increased permeability of the BBB also contributes to increased seizure susceptibility in patients with epilepsy. The endothelial glycocalyx is coating the luminal side of the endothelium and can be considered as the first barrier of the BBB. We hypothesise that an altered glycocalyx thickness plays a role in the aetiology of temporal lobe epilepsy (TLE), the most common type of epilepsy. Here, we propose a protocol that allows intraoperative assessment of the cerebrovascular glycocalyx thickness in patients with TLE and assess whether its thickness is decreased in patients with TLE when compared with controls. METHODS AND ANALYSIS: This protocol is designed as a prospective observational case-control study in patients who undergo resective brain surgery as treatment for TLE. Control subjects are patients without a history of epileptic seizures, who undergo a craniotomy or burr hole surgery for other indications. Intraoperative glycocalyx thickness measurements of sublingual, cortical and hippocampal microcirculation are performed by video microscopy using sidestream dark-field imaging. Demographic details, seizure characteristics, epilepsy risk factors, intraoperative haemodynamic parameters and histopathological evaluation are additionally recorded. ETHICS AND DISSEMINATION: This protocol has been ethically approved by the local medical ethical committee (ID: NL51594.068.14) and complies with the Declaration of Helsinki and principles of Good Clinical Practice. Informed consent is obtained before study enrolment and only coded data will be stored in a secured database, enabling an audit trail. Results will be submitted to international peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: NTR5568.

Oxidized lipoproteins degrade the endothelial surface layer : implications for platelet-endothelial cell adhesion.

BACKGROUND: Flowing erythrocytes and platelets are separated from the luminal endothelial cell (EC) surface by a 0.5-microm-wide space named the endothelial surface layer. We hypothesized that the disruption of the endothelial surface layer by oxidized low-density lipoproteins (Ox-LDL) contributes to atherogenic increases in vascular wall adhesiveness. METHODS AND RESULTS: The hamster cremaster muscle preparation was used for intravital microscopic observation of the distance between erythrocytes and the capillary EC surface. Moderate Ox-LDL was prepared by exposing native LDL to CuSO(4) for 6 hours. The dimension of the EC surface layer averaged 0.6+/-0.1 microm during control situations, but a bolus intravenous injection of Ox-LDL (0.4 mg/100 g of body weight) transiently diminished the EC surface layer by 60% within 25 minutes, which correlated with a transient increase in the number of platelet-EC adhesions. Combined administration of superoxide dismutase and catalase completely blocked the effect of Ox-LDL on the dimension of the EC surface layer and inhibited platelet-EC adhesion. CONCLUSIONS: Oxygen-derived free radicals mediate the disruption of the EC surface layer and increase vascular wall adhesiveness by Ox-LDL.

Prolonged diastolic time fraction protects myocardial perfusion when coronary blood flow is reduced.

BACKGROUND: Because coronary blood flow is impeded during systole, the duration of diastole is an important determinant of myocardial perfusion. The aim of this study was to show that coronary flow modulates the duration of diastole at constant heart rate. METHODS AND RESULTS: In anesthetized, open-chest dogs, diastolic time fraction (DTF) increased significantly when coronary flow was reduced by lowering perfusion pressure from 100 to 70, 55, and 40 mm Hg. On average, DTF increased from 0.47+/-0.04 to 0.55+/-0.03 after a pressure step from 100 to 40 mm Hg in control, from 0.42+/-0.04 to 0.47+/-0.04 after administration of adenosine, and from 0.46+/-0.07 to 0.55+/-0.06 after L-NMMA (mean+/-SD, 6 dogs for control and adenosine, 4 dogs for L-NMMA, all P<0.05). Flow normalized to its value at full dilation and pressure of 90 mm Hg (375+/-25 mL/min) increased during the period of reduced pressure at 40 mm Hg; control, from 0.005+/-63 (2 seconds after pressure step) to 0.09+/-0.06 (15 seconds after pressure step); with adenosine, from 0.19+/-0.06 to 0. 22+/-0.06; and with L-NMMA, from 0.013+/-0.007 to 0.12+/-0.02 (all P<0.05). The increase in DTF at low pressure may be explained by a decrease in interstitial volume at low pressure, which either decreases the preload of the myocytes or reduces the buffer capacity for ions determining repolarization, thereby causing an earlier onset of relaxation. CONCLUSIONS: Because the largest increase in DTF occurs at pressures below the autoregulatory range when blood flow to the subendocardium is closely related to DTF, modulation of DTF by coronary blood flow can provide an important regulatory mechanism to match supply and demand of the myocardium when vasodilatory reserve is exhausted.

Visualisation of intramural coronary vasculature by an imaging cryomicrotome suggests compartmentalisation of myocardial perfusion areas.

A technique is presented for the 3D visualisation of the coronary arterial tree using an imaging cryomicrotome. After the coronary circulation of the excised heart was filled with a fluorescent plastic, the heart was frozen and mounted in the cryomicrotome. The heart was then sliced serially, with a slice thickness of 40 microm, and digital images were taken from each cutting plane of the remaining bulk material using appropriate excitation and emission filters. Using maximum intensity projections over a series of images in the cutting plane and perpendicular plane, the structural organisation of intramural vessels was visualised in the present study. The branching end in the smallest visible vessels, which define tissue areas that are well delineated from each other by 1-2 mm wide bands populated only by vessels less than 40 microm in diameter. The technique presented here allows further quantification in the future of the 3D structure of the coronary arterial tree by image analysis techniques.

Lack of effect of clonidine on stuttering in children.

OBJECTIVE: The authors studied the effects of the alpha 2-receptor agonist clonidine on stuttering in children. METHOD: Using a double-blind crossover study, they gave placebo or 4 micrograms/kg body weight per day to 25 stuttering children who were 6-13 years old. Stuttering was measured by counting the occurrences of four elementary speech difficulties and by asking parents and teachers to give an overall impression of the amount of stuttering, as well as their impression of how troublesome the stuttering was to the children. RESULTS: Clonidine did not improve stuttering. CONCLUSIONS: Clonidine cannot be recommended as a useful drug for treating children who stutter.

Red blood cell velocity in nailfold capillaries during hyperbaric oxygenation.

Microcirculatory hemodynamics of the skin during hyperbaric oxygenation were assessed by determination of nailfold capillary red blood cell velocity (Vrbc). Under hyperbaric conditions a continuous increase in Vrbc was found. Control values, 0.43 +/- 0.12 mm. sec-1 (mean +/- sem), were significantly (P < 0.05) lower compared with Vrbc at the end of hyperbaric oxygenation (0.62 +/- 0.16 mm.sec-1).

Heterogeneous NADH fluorescence during post-anoxic reactive hyperemia in saline perfused rat heart.

In the present study epicardial NADH fluorescence photographs were taken of rat hearts during dynamic transitions of oxygen content of the myocardium. Hearts were perfused in a Langendorff set-up where it was possible to switch between low and high-pO2 perfusates. NADH fluorescence photographs were taken with a suitable fluorescence set-up and photo negatives digitized and analyzed by use of a computer. Restoration of perfusion with a high-pO2 solution resulted in a reactive hyperemic flow being established. Prior to the occlusion being lifted high NADH fluorescence was observed. Reactive hyperemic flow was associated with heterogenic NADH fluorescence patterns which diminished as control flow was restored. The patterns observed during reactive hyperemia were identical to those observed when tissue oxygen was restored by high-pO2 perfusion following high flow hypoxia achieved by low-pO2 perfusion. This study shows that heterogenic epicardial flow patterns are associated with reactive hyperemia.

Degradation of the endothelial glycocalyx is associated with chylomicron leakage in mouse cremaster muscle microcirculation.

A thick endothelial glycocalyx contributes to the barrier function of vascular endothelium in macro- and microcirculation. We hypothesised in the current study that diet-induced hyperlipidaemia perturbs the glycocalyx, resulting in decreased dimensions of this layer and increased transendothelial lipoprotein leakage in capillaries. Glycocalyx thickness was measured in mouse cremaster muscle capillaries by intravital microscopy from the distance between flowing red blood cells and the endothelial surface. In control C57BL/6 mice on standard chow, glycocalyx thickness measured 0.58 ± 0.01 (mean ± SEM) µm, and no lipoproteins were observed in the tissue. After three months administration of an either mild or severe high-fat / high-cholesterol diet (HFC) to C57BL/6 and ApoE3-Leiden mice, circulating large lipoproteins appeared into the subendothelial space in an increasing proportion of cremaster capillaries, and these capillaries displayed reduced glycocalyx dimensions of 0.40 ± 0.02 and 0.30 ± 0.01 µm (C57BL/6 mice), and 0.37 ± 0.01 and 0.28 ± 0.01 µm (ApoE3-Leiden mice), after the mild and severe HFC diet, respectively. The chylomicron nature of the accumulated lipoproteins was confirmed by observations of subendothelial deposition of DiI-labeled chylomicrons in capillaries after inducing acute glycocalyx degradation by heparitinase in normolipidaemic C57BL/6 mice. It is concluded that while under control conditions the endothelial glycocalyx contributes to the vascular barrier against transvascular lipoprotein leakage in the microcirculation, diet-induced hyperlipidaemia reduces the thickness of the glycocalyx, thereby facilitating leakage of chylomicrons across the capillary wall.

Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis.

The endothelial glycocalyx (EG), the luminal cover of endothelial cells, is considered to be atheroprotective. During atherogenesis, platelets adhere to the vessel wall, possibly triggered by simultaneous EG modulation. It was the objective of this study to investigate both EG thickness and platelet-vessel wall interactions during atherogenesis in the same experimental model. Intravital fluorescence microscopy was used to study platelet-vessel wall interactions in vivo in common carotid arteries and bifurcations of C57bl6/J (B6) and apolipoprotein E knock-out (ApoE-/-) mice (age 7 - 31 weeks). At the same locations, EG thickness was determined ex vivo using two-photon laser scanning microscopy. In ApoE-/- bifurcations the overall median level of adhesion was 48 platelets/mm2 (interquartile range: 16 - 80), which was significantly higher than in B6 bifurcations (0 (0 - 16), p = 0.001). This difference appeared to result from a significant age-dependent increase in ApoE-/- mice, while no such change was observed in B6 mice. At the same time, the EG in ApoE-/- bifurcations was significantly thinner than in B6 bifurcations (2.2 vs. 2.5 µm, respectively; p < 0.05). This resulted from the fact that in B6 bifurcations EG thickness increased with age (from 2.4 µm in young mice to 3.0 µm in aged ones), while in bifurcations of ApoE-/- mice this growth appeared to be absent (2.2 µm at all ages). During atherogenesis, platelet adhesion to the wall of the carotid artery bifurcation increases significantly. At the same location, EG growth with age is hampered. Therefore, glycocalyx-reinforcing strategies could possibly ameliorate atherosclerosis.

Tumor necrosis factor-alpha inhibition protects against endotoxin-induced endothelial glycocalyx perturbation.

OBJECTIVE: Inflammatory stimuli profoundly increase the vulnerability of the vessel wall to atherogenesis. The endothelial glycocalyx, a layer of glycosaminoglycans and proteoglycans covering the luminal side of the vasculature, has recently emerged as an orchestrator of vascular homeostasis. In the present study, we investigated whether endotoxin-induced inflammatory reactions lead to a decrease of endothelial glycocalyx thickness in humans and whether tumor necrosis factor-alpha (TNFalpha) plays a role in this process. DESIGN, SUBJECTS AND INTERVENTION: Healthy male volunteers received low-dose endotoxin (1ng/kg) intravenously, with (n=8) or without (n=13) pre-treatment with the soluble TNFalpha receptor etanercept. Endothelial glycocalyx thickness and related parameters were determined after endotoxin challenge. RESULTS: Endotoxin resulted in a profound reduction in microvascular glycocalyx thickness (from 0.60+/-0.1 to 0.30+/-0.1microm, p<0.01). Concomitantly, plasma levels of the principal glycocalyx constituent hyaluronan (62+/-18 to 85+/-24ng/mL, p<0.05), monocyte activation and coagulation activation increased (F1+2; 0.3+/-0.1 to 2.8+/-1.5nmol/L, p<0.05 and d-dimer; from 0.2+/-0.1 to 0.4+/-0.1mg/L, p<0.05 compared to baseline). Inhibition of TNFalpha by etanercept attenuated loss of microvascular glycocalyx thickness (0.54+/-0.1 to 0.35+/-0.1mum, p<0.05). Changes in hyaluronan (58+/-13 to 46+/-10ng/mL, p<0.05) and coagulation activation were also attenuated (F1+2; 0.3+/-0.1 to 2.1+/-0.9nmol/L and d-dimer; from 0.2+/-0.1 to 0.3+/-0.1mg/L, p<0.05 compared to baseline). CONCLUSIONS: These data suggest that inflammatory activity, in part mediated by TNFalpha, leads to perturbation of the endothelial glycocalyx in humans. This may contribute to the vascular vulnerability induced by inflammation.

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

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

Perturbation of hyaluronan metabolism predisposes patients with type 1 diabetes mellitus to atherosclerosis.

AIMS/HYPOTHESIS: Cardiovascular disease contributes to mortality in type 1 diabetes mellitus, but the specific pathophysiological mechanisms remain to be established. We recently showed that the endothelial glycocalyx, a protective layer of proteoglycans covering the endothelium, is severely perturbed in type 1 diabetes, with concomitantly increased plasma levels of hyaluronan and hyaluronidase. In the present study, we evaluated the relationship between hyaluronan and hyaluronidase with carotid intima-media thickness (cIMT), an established surrogate marker for cardiovascular disease. SUBJECTS AND METHODS: Non-smoking type 1 diabetes patients without micro- or macrovascular complications and matched controls were recruited and cIMT of both carotid arteries was measured. To evaluate the relationship between cIMT and hyaluronan and hyaluronidase as well as other parameters, uni- or multivariate regression analyses were performed. RESULTS: We included 99 type 1 diabetes patients (age 10-72 years) and 99 age- and sex-matched controls. Mean cIMT, HbA(1c), high sensitivity C-reactive protein, hyaluronan and hyaluronidase were significantly increased in type 1 diabetes vs controls. Plasma hyaluronan and hyaluronidase were correlated in type 1 diabetes. In univariate regression analyses, mean IMT was associated with plasma hyaluronan, age and male sex, whereas after multivariate analysis only age and sex remained statistically significant. CONCLUSIONS/INTERPRETATION: We conclude that type 1 diabetes patients show structural changes of the arterial wall associated with increased hyaluronan metabolism. These data may lend further support to altered glycosaminoglycan metabolism in type 1 diabetes as a potential mechanism involved in accelerated atherogenesis.

Vasculoprotective properties of the endothelial glycocalyx: effects of fluid shear stress.

The endothelial glycocalyx exerts a wide array of vasculoprotective effects via inhibition of coagulation and leucocyte adhesion, by contributing to the vascular permeability barrier and by mediating shear stress-induced NO release. In this review, we will focus on the relationship 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 hyperglycaemia on whole body glycocalyx volume in humans and its effect on endothelial function.

Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries.

A thick endothelial surface coat consisting of the glycocalyx and associated plasma proteins has been hypothesized to reduce functional capillary volume available for flowing plasma macromolecules and blood cells. The purpose of this study was to compare anatomic and functional capillary diameters available for macromolecules, RBCs, and WBCs in hamster cremaster muscle capillaries. Bright-field and fluorescence microscopy provided similar estimates (mean +/- SE) of the anatomic capillary diameter: 5.1 +/- 0.1 microns (bright field, 39 capillaries in 10 animals) and 5.1 +/- 0.2 microns (membrane dye PKH26, 18 capillaries in 2 animals). Estimates of functional diameters were obtained by measuring the width of RBCs and WBCs and the intracapillary distribution of systemically injected fluorescein isothiocyanate (FITC)-dextran 70. WBCs (5.1 +/- 0.2 microns) fully occupied the anatomic capillary cross section. In contrast, the widths of RBCs (3.9 +/- 0.2 microns, 21 capillaries in 8 animals) and FITC-dextran (4.3 +/- 0.2 microns, 21 capillaries in 8 animals) were significantly smaller than the anatomic capillary diameter. Continuous (1- to 5-minute) excitation of fluorochromes in the capillary lumen (light-dye treatment) increased the width of RBCs passing the treated site from 3.6 +/- 0.3 to 4.4 +/- 0.3 microns (6 capillaries in 4 animals) and the width of the FITC-dextran column from 4.1 +/- 0.2 to 4.6 +/- 0.3 microns (10 capillaries in 7 animals). Furthermore, light-dye treatment increased capillary tube hematocrit by 60% in 40-microns-long capillary segments compared with untreated sites in the same capillaries. It is concluded that the wall of skeletal muscle capillaries is decorated with a 0.4- to 0.5-microns-thick endothelial surface coat, which may represent the true active interface between blood and the capillary wall.

Capillary endothelial surface layer selectively reduces plasma solute distribution volume.

We previously reported that a 0.4- to 0.5-microm-thick endothelial surface layer confines Dextran 70 (70 kDa) to the central core of hamster cremaster muscle capillaries. In the present study we used a variety of plasma tracers to probe the barrier properties of the endothelial surface layer using combined fluorescence and brightfield intravital microscopy. No permeation of the endothelial surface layer was observed for either neutral or anionic dextrans >/=70 kDa, but a neutral Dextran 40 (40 kDa) and neutral free dye (rhodamine, 0.4 kDa) equilibrated with the endothelial surface layer within 1 min. In contrast, small anionic tracers of similar size (0. 4-40 kDa) permeated the endothelial surface layer relatively slowly with half-times (tau(50)) between 11 and 60 min, depending on tracer size. Furthermore, two plasma proteins, fibrinogen (340 kDa) and albumin (67 kDa), moved slowly into the endothelial surface layer at the same rates, despite greatly differing sizes (tau(50) approximately 40 min). Dextran 70, which did not enter the glycocalyx over the course of these experiments, entered at the same rate as free albumin when it was conjugated to albumin. These findings demonstrate that for anionic molecules size and charge have a profound effect on the penetration rate into the glycocalyx. The equal rates of penetration of the glycocalyx demonstrated by the different protein molecules suggests that multiple factors may influence the penetration of the barrier, including molecular size, charge, and structure.