Bart Eskens

Association of kidney function with changes in the endothelial surface layer.

BACKGROUND AND OBJECTIVES ESRD is accompanied by endothelial dysfunction. Because the endothelial glycocalyx (endothelial surface layer) governs interactions between flowing blood and the vessel wall, perturbation could influence disease progression. This study used a novel noninvasive sidestream-darkfield imaging method, which measures the accessibility of red blood cells to the endothelial surface layer in the microcirculation (perfused boundary region), to investigate whether renal function is associated with endothelial surface layer dimensions. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Perfused boundary region was measured in control participants (n=10), patients with ESRD (n=23), participants with normal kidney function after successful living donor kidney transplantation (n=12), and patients who developed interstitial fibrosis/tubular atrophy after kidney transplantation (n=10). In addition, the endothelial activation marker angiopoietin-2 and shed endothelial surface layer components syndecan-1 and soluble thrombomodulin were measured using ELISA. RESULTS Compared with healthy controls (1.82 ± 0.16 µm), ESRD patients had a larger perfused boundary region (+0.23; 95% confidence interval, 0.46 to <0.01; P<0.05), which signifies loss of endothelial surface layer dimensions. This large perfused boundary region was accompanied by higher circulating levels of syndecan-1 (+57.71; 95% confidence interval, 17.38 to 98.04; P<0.01) and soluble thrombomodulin (+12.88; 95% confidence interval, 0.29 to 25.46; P<0.001). After successful transplantation, the perfused boundary region was indistinguishable from healthy controls (without elevated levels of soluble thrombomodulin or syndecan-1). In contrast, however, patients who developed interstitial fibrosis and tubular atrophy showed a large perfused boundary region (+0.36; 95% confidence interval, 0.09 to 0.63; P<0.01) and higher levels of endothelial activation markers. In addition, a significant correlation between perfused boundary region, angiopoietin-2, and eGFR was observed (perfused boundary region versus GFR: Spearmans ρ=0.31; P<0.05; perfused boundary region versus angiopoietin-2: Spearmans ρ=-0.33; P<0.05). CONCLUSION Reduced renal function is strongly associated with low endothelial surface layer dimensions. After successful kidney transplantation, the endothelial surface layer is indistinguishable from control.

Rapid Insulin-Mediated Increase in Microvascular Glycocalyx Accessibility in Skeletal Muscle May Contribute to Insulin-Mediated Glucose Disposal in Rats

It has been demonstrated that insulin-mediated recruitment of microvascular blood volume is associated with insulin sensitivity. We hypothesize that insulin rapidly stimulates penetration of red blood cells (RBC) and plasma into the glycocalyx and thereby promotes insulin-mediated glucose uptake by increasing intracapillary blood volume. Experiments were performed in rats; the role of the glycocalyx was assessed by enzymatic degradation using a bolus of hyaluronidase. First, the effect of insulin on glycocalyx accessibility was assessed by measuring the depth of penetration of RBCs into the glycocalyx in microvessels of the gastrocnemius muscle with Sidestream Dark-field imaging. Secondly, peripheral insulin sensitivity was determined using intravenous insulin tolerance tests (IVITT). In addition, in a smaller set of experiments, intravital microscopy of capillary hemodynamics in cremaster muscle and histological analysis of the distribution of fluorescently labeled 40 kDa dextrans (D40) in hindlimb muscle was used to evaluate insulin-mediated increases in capillary blood volume. Insulin increased glycocalyx penetration of RBCs by 0.34±0.44 µm (P<0.05) within 10 minutes, and this effect of insulin was greatly impaired in hyaluronidase treated rats. Further, hyaluronidase treated rats showed a 35±25% reduction in whole-body insulin-mediated glucose disposal compared to control rats. Insulin-mediated increases in capillary blood volume were reflected by a rapid increase in capillary tube hematocrit from 21.1±10.1% to 29.0±9.8% (P<0.05), and an increase in D40 intensity in individual capillaries of 134±138% compared to baseline at the end of the IVITT. These effects of insulin were virtually abolished in hyaluronidase treated animals. In conclusion, insulin rapidly increases glycocalyx accessibility for circulating blood in muscle, and this is associated with an increased blood volume in individual capillaries. Hyaluronidase treatment of the glycocalyx abolishes the effects of insulin on capillary blood volume and impairs insulin-mediated glucose disposal.

Early impairment of skeletal muscle endothelial glycocalyx barrier properties in diet-induced obesity in mice.

While previous studies have indicated an important role for the endothelial glycocalyx in regulation of microvascular function, it was recently shown that acute enzymatic glycocalyx degradation in rats was associated with an impaired insulin‐mediated glucose disposal. The aim of this study was to determine whether glycocalyx damage in skeletal muscle occurs at an early stage of diet‐induced obesity (DIO). The microcirculation of the hindlimb muscle of anesthetized C57Bl/6 mice, fed chow (CON) or a high‐fat diet (HFD) for 6 and 18 weeks (w), respectively, was visualized with a Sidestream Dark‐Field camera, and glycocalyx barrier properties were derived from the calculated perfused boundary region (PBR). Subsequently, an intraperitoneal glucose tolerance test was performed and the area under the curve (AUC) of blood glucose was calculated. Impairment of glycocalyx barrier properties was already apparent after 6 weeks of HFD and remained after 18 weeks of HFD (PBR [in μm]: 0.81 ± 0.03 in CON_6w vs. 0.97 ± 0.04 in HFD_6w and 1.02 ± 0.07 in HFD_18w [both P < 0.05]). Glucose intolerance appeared to develop more slowly (AUC [in mmol/L × 120 min]: 989 ± 61 in CON_6w vs. 1204 ± 89 in HFD_6w [P = 0.11] and 1468 ± 84 in HFD_18w [P < 0.05]) than the impairment of glycocalyx barrier properties. The data indicate that damage to the endothelial glycocalyx is an early event in DIO. It is suggested that glycocalyx damage may contribute to the development of insulin resistance in obesity.

Potential role of cytochrome P450-1B1 in the metabolic activation of 4-aminobiphenyl in humans

Metabolites of the human carcinogen 4‐aminobiphenyl (4‐ABP) form hemoglobin (Hb) adducts, which represent a useful biomarker for exposure. However, not every individual responds to a similar degree to 4‐ABP exposure, and variations in 4‐ABP‐Hb adduct formation might be explained by genetic polymorphisms in genes coding for enzymes involved in 4‐ABP metabolism. 4‐ABP‐Hb adducts were measured in blood samples from 57 smoking and 10 non‐smoking volunteers. An association was found between cigarette smoking and 4‐ABP‐Hb adduct levels in smokers (R2 = 0.5, P < 0.001). Subsequently, subjects were genotyped for 12 polymorphisms in seven genes involved in biotransformation reactions. From this selection of polymorphisms, a significant impact was found for the CYP1B1 Leu432Val polymorphism (P = 0.021), which has been reported to lead to a decrease in enzyme activity. Indeed higher levels of 4‐ABP‐Hb adducts were observed in homo‐ and heterozygous carriers of the CYP1B1 432Leu as compared to the double CYP1B1 432Val genotype. A significant interaction between these CYP1B1 genotypes and the level of exposure was found (P = 0.003). Noteworthy, a saturation effect was observed for 4‐ABP‐Hb adduct formation at high smoking doses limited to carriers of the CYP1B1 432Leu allele. No effect of polymorphisms in other genes were found. This is the first study in humans suggesting a crucial role of the CYP1B1 enzyme in 4‐ABP metabolism, indicating a protective effect of the CYP1B1 Leu432Val polymorphism against the formation of 4‐ABP‐Hb adduct levels, depending on the smoking dose.

Acute enzymatic glycocalyx degradation results in reduced insulin sensitivity but normal glucose tolerance in conscious rats

Perturbation of the endothelial glycocalyx has been suggested to be an early event in the development of endothelial dysfunction during increased cardiovascular risk exposure. Previously, we showed in anesthetized rats that acute enzymatic degradation of the endothelial glycocalyx was associated with impaired insulin-mediated glucose disposal, suggesting a role of the glycocalyx in regulation of glucose homeostasis. In the current study, we tested the acute effect of enzymatic glycocalyx treatment on glucose tolerance and insulin sensitivity in conscious rats. In rats that had had their femoral artery and vein cannulated 1 week before the experiment, intravenous glucose and insulin tolerance tests were performed before and after acute degradation of the endothelial glycocalyx using an intravenous bolus of hyaluronidase. During the intravenous glucose tolerance test, glucose tolerance did not differ between the hyaluronidase-treated and control animals, but was associated with a 1.5-fold higher plasma insulin level in the former group. Further, glycocalyx treatment significantly decreased insulin-mediated glucose disposal during intravenous insulin tolerance tests, as observed from the reduced insulin-mediated glucose disposal rate from 3.3±0.1%/min in control rats to 2.6±0.2%/min in the hyaluronidase-treated rats. These results support a role for the endothelial glycocalyx in the regulation of peripheral insulin sensitivity. The reduction in insulin sensitivity during acute glycocalyx damage, however, does not have an immediate effect on glucose tolerance because of a compensatory increase in plasma insulin levels.

SPARC preserves endothelial glycocalyx integrity, and protects against adverse cardiac inflammation and injury during viral myocarditis

Myocardial damage as a consequence of cardiotropic viruses leads to a broad variety of clinical presentations and is still a complicated condition to diagnose and treat. Whereas the extracellular matrix protein Secreted Protein Acidic and Rich in Cysteine or SPARC has been implicated in hypertensive and ischemic heart disease by modulating collagen production and cross-linking, its role in cardiac inflammation and endothelial function is yet unknown. Absence of SPARC in mice resulted in increased cardiac inflammation and mortality, and reduced cardiac systolic function upon coxsackievirus-B3 induced myocarditis. Intra-vital microscopic imaging of the microvasculature of the cremaster muscle combined with electron microscopic imaging of the microvasculature of the cardiac muscle uncovered the significance of SPARC in maintaining endothelial glycocalyx integrity and subsequent barrier properties to stop inflammation. Moreover, systemic administration of recombinant SPARC restored the endothelial glycocalyx and consequently reversed the increase in inflammation and mortality observed in SPARC KO mice in response to viral exposure. Reducing the glycocalyx in vivo by systemic administration of hyaluronidase, an enzyme that degrades the endothelial glycocalyx, mimicked the barrier defects found in SPARC KO mice, which could be restored by subsequent administration of recombinant SPARC. In conclusion, the secreted glycoprotein SPARC protects against adverse cardiac inflammation and mortality by improving the glycocalyx function and resulting endothelial barrier function during viral myocarditis.