Hans L. Mooij

Hyperglycemia is associated with increased susceptibility to atherothrombotic stimuli. The glycocalyx, a layer of proteoglycans covering the endothelium, is involved in the protective capacity of the vessel wall. We therefore evaluated whether hyperglycemia affects the glycocalyx, thereby increasing vascular vulnerability. The systemic glycocalyx volume was estimated by comparing the distribution volume of a glycocalyx permeable tracer (dextran 40) with that of a glycocalyx impermeable tracer (labeled erythrocytes) in 10 healthy male subjects. Measurements were performed in random order on five occasions: two control measurements, two measurements during normoinsulinemic hyperglycemia with or without N-acetylcysteine (NAC) infusion, and one during mannitol infusion. Glycocalyx measurements were reproducible (1.7 +/- 0.2 vs. 1.7 +/- 0.3 l). Hyperglycemia reduced glycocalyx volume (to 0.8 +/- 0.2 l; P < 0.05), and NAC was able to prevent the reduction (1.4 +/- 0.2 l). Mannitol infusion had no effect on glycocalyx volume (1.6 +/- 0.1 l). Hyperglycemia resulted in endothelial dysfunction, increased plasma hyaluronan levels (from 70 +/- 6 to 112 +/- 16 ng/ml; P < 0.05) and coagulation activation (prothrombin activation fragment 1 + 2: from 0.4 +/- 0.1 to 1.1 +/- 0.2 nmol/l; d-dimer: from 0.27 +/- 0.1 to 0.55 +/- 0.2 g/l; P < 0.05). Taken together, these data indicate a potential role for glycocalyx perturbation in mediating vascular dysfunction during hyperglycemia.

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 glycocalyx-permeable tracer (dextran 40) to that of a glycocalyx-impermeable 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 microm, 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.

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).

The endothelial glycocalyx is increasingly considered as an intravascular compartment that protects the vessel wall against pathogenic insults. The purpose of this study was to translate an established experimental method of estimating capillary glycocalyx dimension into a clinically useful tool and to assess its reproducibility in humans. We first evaluated by intravital microscopy the relation between the distance between the endothelium and erythrocytes, as a measure of glycocalyx thickness, and the transient widening of the erythrocyte column on glycocalyx compression by passing leukocytes in hamster cremaster muscle capillaries. We subsequently assessed sublingual microvascular glycocalyx thickness in 24 healthy men using orthogonal polarization spectral imaging. In parallel, systemic glycocalyx volume (using a previously published tracer dilution technique) as well as cardiovascular risk profiles were assessed. Estimates of microvascular glycocalyx dimension from the transient erythrocyte widening correlated well with the size of the erythrocyte-endothelium gap (r = 0.63). Measurements in humans were reproducible (0.58 +/- 0.16 and 0.53 +/- 0.15 microm, coefficient of variance 15 +/- 5%). In univariate analysis, microvascular glycocalyx thickness significantly correlated with systemic glycocalyx volume (r = 0.45), fasting plasma glucose (r = 0.43), and high-density lipoprotein-cholesterol (r = 0.40) and correlated negatively with low-density lipoprotein-cholesterol (r = -0.41) as well as body mass index (r = -0.45) (all P < 0.05). In conclusion, the dimension of the endothelial glycocalyx can be measured reproducibly in humans and is related to cardiovascular risk factors. It remains to be tested whether glycocalyx dimension can be used as an early marker of vascular damage and whether therapies aimed at glycocalyx repair can protect the vasculature against pathogenic challenges.