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Background The endothelial glycocalyx (eGC) covers the luminal surface of the vascular endothelium and plays an important protective role in systemic inflammatory states and particularly in sepsis. Its breakdown leads to capillary leak and organ dysfunction. Moreover, sepsis-induced alterations of sublingual microcirculation are associated with a worse clinical outcome. The present study was performed to investigate the associations between eGC dimensions and established parameters of microcirculation dysfunction in sepsis. Methods This observational, prospective, cross-sectional study included 40 participants, of which 30 critically ill septic patients were recruited from intensive care units of a university hospital and 10 healthy volunteers served as controls. The established microcirculation parameters were obtained sublingually and analyzed according to the current recommendations. In addition, the perfused boundary region (PBR), an inverse parameter of the eGC dimensions, was measured sublingually, using novel data acquisition and analysis software (GlycoCheck™). Moreover, we exposed living endothelial cells to 5% serum from a subgroup of study participants, and the delta eGC breakdown, measured with atomic force microscopy (AFM), was correlated with the paired PBR values. Results In septic patients, sublingual microcirculation was impaired, as indicated by a reduced microvascular flow index (MFI) and a reduced proportion of perfused vessels (PPV) compared to those in healthy controls (MFI, 2.93 vs 2.74, p  = 0.002; PPV, 98.53 vs 92.58, p  = 0.0004). PBR values were significantly higher in septic patients compared to those in healthy controls, indicating damage of the eGC (2.04 vs 2.34, p  < 0.0001). The in vitro AFM data correlated exceptionally well with paired PBR values obtained at the bedside (rs = − 0.94, p  = 0.02). Both PBR values and microcirculation parameters correlated well with the markers of critical illness. Interestingly, no association was observed between the PBR values and established microcirculation parameters. Conclusion Our findings suggest that eGC damage can occur independently of microcirculatory impairment as measured by classical consensus parameters. Further studies in critically ill patients are needed to unravel the relationship of glycocalyx damage and microvascular impairment, as well as their prognostic and therapeutic importance in sepsis. Trial registration Retrospectively registered: Clinicaltrials.gov, NCT03960307

Background The availability of handheld, noninvasive sublingual video-microscopes allows for visualization of the microcirculation in critically ill patients. Recent studies demonstrate that reduced numbers of blood-perfused microvessels and increased penetration of erythrocytes into the endothelial glycocalyx are essential components of microvascular dysfunction. The aim of this study was to identify novel microvascular variables to determine the level of microvascular dysfunction in sepsis and its relationship with clinical variables. Methods This observational, prospective, cross-sectional study included 51 participants, of which 34 critically ill sepsis patients were recruited from intensive care units of a university hospital. Seventeen healthy volunteers served as controls. All participants underwent sublingual videomicroscopy by sidestream darkfield imaging. A new developed version of the Glycocheck™ software was used to quantify vascular density, perfused boundary region (PBR-an inverse variable of endothelial glycocalyx dimensions), red blood cell (RBC) velocity, RBC content, and blood flow in sublingual microvessels with diameters between 4 and 25 µm. Results A detailed analysis of adjacent diameter classes (1 µm each) of vessels between 4 and 25 µm revealed a severe reduction of vascular density in very small capillaries (5–7 µm), which correlated with markers of sepsis severity. Analysis of RBC velocity (V RBC ) revealed a strong dependency between capillary and feed vessel V RBC in sepsis patients ( R 2  = 0.63, p  < 0.0001) but not in healthy controls ( R 2  = 0.04, p  = 0.43), indicating impaired capillary (de-)recruitment in sepsis. This finding enabled the calculation of capillary recruitment and dynamic capillary blood volume (CBV dynamic ). Moreover, adjustment of PBR to feed vessel V RBC further improved discrimination between sepsis patients and controls by about 50%. By combining these dynamic microvascular and glycocalyx variables, we developed the microvascular health score (MVHS dynamic ™), which decreased from 7.4 [4.6–8.7] in controls to 1.8 [1.4–2.7] in sepsis patients ( p  < 0.0001) and correlated with sepsis severity. Conclusion We introduce new important diameter-specific quantification and differentiated analysis of RBC kinetics, a key to understand microvascular dysfunction in sepsis. MVHS dynamic , which has a broad bandwidth to detect microvascular (dys-) function, might serve as a valuable tool to detect microvascular impairment in critically ill patients.

RationalePre-clinical and autopsy studies have fueled the hypothesis that a dysregulated vascular endothelium might play a central role in the pathogenesis of ARDS and multi-organ failure in COVID-19.ObjectivesTo comprehensively characterize and quantify microvascular alterations in patients with COVID-19.Methods Hospitalized adult patients with moderate-to-severe or critical COVID-19 (n = 23) were enrolled non-consecutively in this prospective, observational, cross-sectional, multi-center study. Fifteen healthy volunteers served as controls. All participants underwent intravital microscopy by sidestream dark field imaging to quantify vascular density, red blood cell velocity (VRBC), and glycocalyx dimensions (perfused boundary region, PBR) in sublingual microvessels. Circulating levels of endothelial and glycocalyx-associated markers were measured by multiplex proximity extension assay and enzyme-linked immunosorbent assay.Measurements and main resultsCOVID-19 patients showed an up to 90% reduction in vascular density, almost exclusively limited to small capillaries (diameter 4–6 µm), and also significant reductions of VRBC. Especially, patients on mechanical ventilation showed severe glycocalyx damage as indicated by higher PBR values (i.e., thinner glycocalyx) and increased blood levels of shed glycocalyx constituents. Several markers of endothelial dysfunction were increased and correlated with disease severity in COVID-19. PBR (AUC 0.75, p = 0.01), ADAMTS13 (von Willebrand factor-cleaving protease; AUC 0.74, p = 0.02), and vascular endothelial growth factor A (VEGF-A; AUC 0.73, p = 0.04) showed the best discriminatory ability to predict 60-day in-hospital mortality.ConclusionsOur data clearly show severe alterations of the microcirculation and the endothelial glycocalyx in patients with COVID-19. Future therapeutic approaches should consider the importance of systemic vascular involvement in COVID-19.

Background Deterioration of the endothelial glycocalyx (eGC), a protective carbohydrate-rich layer lining the luminal surface of the endothelium, plays a key role in vascular barrier dysfunction and eventually organ-failure in systemic inflammatory response syndrome and sepsis. Early detection of glycocalyx damage could thus become an important goal in critical care. This study was designed to determine the feasibility and reproducibility of quantitative, real-time glycocalyx measurements performed at bedside in the emergency room (ER) and intensive care unit (ICU). Methods The observational study included 70 patients admitted to the ER or ICU of a university hospital. A physician and the nurse in charge of the patient performed sublingual microcirculatory measurements using sidestream dark field (SDF) imaging. A novel data acquisition and analysis software (GlycoCheck™) was used to analyze the perfused boundary region (PBR), an inverse parameter of endothelial glycocalyx dimensions in vessels with diameters of between 5 and 25 μm. Results The method showed a good intra-observer reproducibility. Specifically, intraclass correlation coefficient analysis showed an excellent reproducibility between the physician’s measurements (0.77 [CI 95%: 0.52–0.89]). The bias between the two PBRs was − 0.077 ± 0.24 μm. Moreover, there were no significant differences in the PBR values obtained by the nurses when compared to those reported by the physician (regarded as the “gold standard” measurement). Intraclass correlation coefficient analysis showed excellent reproducibility between the nurses’ and physician’s PBRs (0.75 [95% CI: 0.52–0.87]). The mean difference between the two PBRs (i.e., the bias) was 0.007 ± 0.25 μm. The nurses’ PBR assessment had a 90% sensitivity (95% CI: 60–99%) and 90% specificity (95% CI: 80–93%) to identify a severely impaired glycocalyx. Conclusion Glycocalyx dimensions can be measured at patients’ bedside precisely by non-invasive assessment of the PBR. This assessment could become part of standard monitoring and contribute to clinical decision-making and resuscitation protocols in clinical trials and daily practice.

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Changes in endothelial glycocalyx are one of the earliest changes in development of cardiovascular disease. The endothelial glycocalyx is both an important biological modifier of interactions between flowing blood and the vessel wall, and a determinant of organ perfusion. We hypothesize that deeper penetration of erythrocytes into the glycocalyx is associated with reduced microvascular perfusion. The population-based prospective cohort study (the Netherlands Epidemiology of Obesity [NEO] study) includes 6,673 middle-aged individuals (oversampling of overweight and obese individuals). Within this cohort, we have imaged the sublingual microvasculature of 915 participants using sidestream darkfield (SDF) imaging together with a recently developed automated acquisition and analysis approach. Presence of RBC (as a marker of microvascular perfusion) and perfused boundary region (PBR), a marker for endothelial glycocalyx barrier properties for RBC accessibility, were assessed in vessels between 5 and 25 µm RBC column width. A wide range of variability in PBR measurements, with a mean PBR of 2.14 µm (range: 1.43-2.86 µm), was observed. Linear regression analysis showed a marked association between PBR and microvascular perfusion, reflected by RBC filling percentage (regression coefficient β: -0.034; 95% confidence interval: -0.037 to -0.031). We conclude that microvascular beds with a thick (\"healthy\") glycocalyx (low PBR), reflects efficient perfusion of the microvascular bed. In contrast, a thin (\"risk\") glycocalyx (high PBR) is associated with a less efficient and defective microvascular perfusion.

In this inaugural paper, we shall provide an overview of the endothelial surface layer or glycocalyx in several roles: as a transport barrier, as a porous hydrodynamic interface in the motion of red and white cells in microvessels, and as a mechanotransducer of fluid shearing stresses to the actin cortical cytoskeleton of the endothelial cell. These functions will be examined from a new perspective, the quasiperiodic ultrastructural model proposed in Squire et al. [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239-255] for the 3D organization of the endothelial surface layer and its linkage to the submembranous scaffold. We shall show that the core proteins in the bush-like structures comprising the matrix have a flexural rigidity, EI, that is sufficiently stiff to serve as a molecular filter for plasma proteins and as an exquisitely designed transducer of fluid shearing stresses. However, EI is inadequate to prevent the buckling of these protein structures during the intermittent motion of red cells or the penetration of white cell microvilli. In these cellular interactions, the viscous draining resistance of the matrix is essential for preventing adhesive molecular interactions between proteins in the endothelial membrane and circulating cellular components.

Background: Cerebral small vessel disease is thought to result from endothelial dysfunction. The glycocalyx, lining the endothelium, is a major determinant of endothelial function. The glycocalyx is partially accessible to flowing red blood cells at its luminal side, called the perfused boundary region (PBR). Glycocalyx damage results in increased PBR, which can be measured in the sublingual microvasculature. We tested whether PBR is increased in patients with cerebral small vessel disease, i.e. lacunar stroke patients, and further distinguished patients with presence of white matter lesions as a sign of extensive cerebral small vessel disease. Methods: We used sidestream dark field imaging of the sublingual microcirculation in 31 lacunar stroke patients (6 with and 25 without white matter lesions) and 19 healthy controls. In each subject, automatic image analyzing software analyzed PBR in more than 3,000 vessel segments and the average dimension of glycocalyx PBR was determined in vessel segments with a diameter of 5-25 μm. Results: PBR did not differ between lacunar stroke patients and healthy controls (2.10 ± 0.25 vs. 2.08 ± 0.24 μm, p = 0.8). However, lacunar stroke patients with white matter lesions had an increased PBR compared with both healthy controls (2.35 ± 0.23 vs. 2.08 ± 0.24 μm, p = 0.03) and patients without white matter lesions (2.04 ± 0.22 μm, p = 0.004). Conclusions: White matter lesions are associated with an increase in the red blood cell permeable part of the sublingual microvascular glycocalyx in lacunar stroke patients. This implicates compromised glycocalyx barrier properties, which is consistent with impaired endothelial function in lacunar stroke patients with white matter lesions.

Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Loss of Endothelial Glycocalyx During Acute Hyperglycemia Coincides With Endothelial Dysfunction and Coagulation Activation In Vivo Max Nieuwdorp 1 , Timon W. van Haeften 2 , Mirella C.L.G. Gouverneur 3 , Hans L. Mooij 1 , Miriam H.P. van Lieshout 1 , Marcel Levi 4 , Joost C.M. Meijers 1 , Frits Holleman 4 , Joost B.L. Hoekstra 4 , Hans Vink 3 , John J.P. Kastelein 1 and Erik S.G. Stroes 1 1 Department of Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands 2 Department of Internal Medicine, University Medical Center Utrecht, Utrecht, the Netherlands 3 Department of Medical Physics, Academic Medical Center, Amsterdam, the Netherlands 4 Department of Internal Medicine, Academic Medical Center, Amsterdam, the Netherlands Address correspondence and reprint requests to Erik S.G. Stroes, MD, PhD, Department of Vascular Medicine, Academic Medical Center, Meibergdreef 9, Rm. F4-159.2, 1105 AZ, Amsterdam, Netherlands. E-mail: e.s.stroes{at}amc.uva.nl Abstract 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. FMD, flow-mediated dilation NAC, N-acetylcysteine Footnotes Accepted October 25, 2005. Received August 24, 2005. DIABETES