The effect of perturbations of the glycocalyx on microvascular perfusion in the obese trauma population: an in vitro study

ObjectivesPatients with morbid obesity have impaired responses to resuscitation following severe injury, which may contribute to adverse outcomes. Obesity is associated with microvascular dysfunction and metabolic changes associated with altered hemorheological profiles. These include decreased red blood cell (RBC) deformity associated with increased aggregation and adhesion. These RBC changes may be impacted by the glycocalyx layer of the endothelial cell (EC) and RBC. Degradation of either or both glycocalyx layers may impair microvascular perfusion. This was studied from blood obtained from patients with obesity and in an in vitro microfluidic device to mimic the microvascular environment.MethodsRBCs were obtained from fresh whole blood from normal controls and patients with obesity (body mass index 37.6–60.0). RBC glycocalyx was indexed by fluorescent intensity and shedding of EC glycocalyx components into the serum was determined by measurement of syndecan-1 and hyaluronic acid. In a second set of experiments, human umbilical vein endothelial cell monolayers (HUVEC) were perfused with RBC suspensions from control and patients with obesity using a microfluidic device and RBC adherence under normoxic or shock conditions (hypoxia+epinephrine) was determined using confocal microscopy. HUVEC glycocalyx thickness and shedding were also measured.ResultsMicrofluidic studies demonstrated that RBC obtained from subjects with obesity had increased adhesion to the endothelial layer, which was more profound under shock conditions versus normal subjects. This appeared to be related to increased shedding of the endothelial glycocalyx following shock as well as a diminished RBC glycocalyx layer in the obese population.ConclusionBlood from patients with obesity have decreased RBC glycocalyx thickness accompanied by evidence of increased EC glycocalyx shedding. In vitro adhesion to the endothelium was more pronounced with RBC from patients with obesity and was significantly greater under ‘shock conditions’. Hemorheological properties of RBC from patients with obesity may account for failure of standard resuscitation procedures in the trauma patient.Level of evidence

Association of endothelial glycocalyx integrity and microvascular perfusion with novel echocardiographic markers and carotid intima-media thickness in patients with psoriasis

Funding Acknowledgements Type of funding sources: None. Introduction Psoriasis has been associated with vascular and myocardial dysfunction through mechanisms of inflammation and oxidative stress. Purpose We aimed to evaluate sublingual microvascular perfusion and glycocalyx barrier properties in psoriasis patients, as well as their correlation with coronary microcirculatory function, markers of myocardial deformation and atherosclerosis (carotid intima-media thickness, IMT). Methods We examined 241 patients with psoriasis and 160 controls, adjusted for age, sex, BMI, smoking, LV mass, heart rate, hyperlipidemia, and office SBP. Perfusion boundary region (PBR), a marker of glycocalyx barrier function, was measured non-invasively in sublingual microvessels with a diameter ranging from 5-25 µm using a dedicated camera (Side field dark imaging, Micrsoscan, Nedelrands). Increased PBR indicates reduced glycocalyx thickness. Indexes of microvascular perfusion, including red blood cell (RBC) filling percentage and functional microvascular density, were also calculated. We measured coronary flow reserve (CFR), carotid IMT and markers of myocardial deformation by speckle tracking imaging utilizing echocardiography [peak twisting, the percentage changes between peak twisting, and untwisting at mitral valve opening (%dpTw – UtwMVO), at peak (%dpTw – UtwPEF), and the end of early LV diastolic filling (%dpTw – UtwEDF)]. Results Psoriasis patients had higher PBR (PBR5-25: 2.131 ± 0.296 vs. 1.769 ± 0.306; PBR5-9: 1.183 ± 0.150 vs. 1.051 ± 0.132; PBR10-19: 2.318 ± 0.581 vs. 1.993 ± 0.365; PBR20-25: 2.650 ± 0.461 vs. 2.269 ± 0.492, all p < 0.05) and impaired LV twisting-untwisting markers compared to controls (pTw: 14.8 ± 7.8 vs. 13.9 ± 3.5; UtwMVO: 10.3 ± 7.3 vs. 9 ± 4.3; %dpTw – UtwMVO: 31 ± 4.1 vs. 38 ± 7; pTwVel: 105 ± 83 vs. 89 ± 21; pUtwVel: -100 ± 53 vs. -93 ± 31, all p < 0.05). In psoriatic population, levels of PBR5-25 and PBR10-19 were inversely correlated to CFR (r=-0.15 and r=-0.17). The first one was also reversely related to peak LV untwisting at aortic valve closure (r=-0.14). Increased levels of PBR5-9 were associated with reduced untwisting at end of the mitral inflow E wave (UtwEDF, r=-0.13) and reduced percentage difference between peak twisting and untwisting at MVO (%dpTw-UntwMVO) (r=-0.15). Furthermore, decreased RBC filling percentage and perfused microvascular density were related to worse LV longitudinal strain (4-chamber) (r=-0.15), and increased IMT (r=-0.14). Finally, a positive correlation between perfused microvascular density and %dpTw-UntwMVO was observed in patients with psoriasis (r = 0.14). All correlations were statistically significant (p < 0.05). Conclusion Endothelial glycocalyx thickness is reduced in patients with psoriasis and is associated with impaired coronary and myocardial function, and vascular atherosclerosis.

The predictive role of endothelial glycocalyx in primary prevention of cardiovascular events:a 6 year follow-up study

Introduction Endothelial glycocalyx is a layer of glycoproteins on endothelial cells preventing vascular wall permeability to circulating proteins and blood cells. Perfused Boundary Region (PBR) of arterial microvessel is a non-invasive marker of endothelial glycocalyx thickness, as increased PBR indicates a reduced endothelial glycocalyx thickness. Cardiovasular risk factors and cardiovascular diseases have been associated with impaired PBR. However, PBR has not been studied as a predictor of major adverse cardiovascular events (MACE) in primary prevention. Methods We studied 400 adults, without history cardiovascular disease. We measured PBR of sublingual vessels with diameter 5–25μm by GlycoCheck Software using a dedicated camera (Sidefield Darkview Imaging, Microscan MV, Nederlands). We recorded a full medical history for each subject. The participants were followed up for a period of 6 years for major cardiovascular events (including Acute Coronary Syndrome, Stroke, decompensated acute heart failure, cardiovascular death and death of all causes). Results The mean age of participants was 49±10 years, 52% were males, 65% active smokers, 33.3% had hyperlipidemia, 12.8% type 2 Diabetes respectively, 30% hypertension and 20.5% had positive family history of premature coronary artery disease. The median PBR value was equal to 1.15 μm. After 6 years of follow up, 42 MACE were recorded (7 cardiovascular deaths). In univariate analysis, subjects with PBR values >1.15μm had greater risk of adverse events compared with those that had lower PBR (relative risk RR=2.83, p<0.05). Diabetes and hypertension were also univariate predictors of MACE (RR=3.71 and RR=2.51, p<0.05). In multivariate analysis after adjusting for atherosclerotic risk factors including diabetes and hypertension, PBR remained a an independent predictor of MACE (RR=2.67, p<0.05) Conclusion Endothelial glycocalyx thickness appears to be an important independent predictor of adverse cardiovascular outcomes. Predictive value endothelial glycocalyx Funding Acknowledgement Type of funding source: None

Decreased endothelial glycocalyx thickness is an early predictor of mortality in sepsis

Microcirculatory alterations play an important role in the early phase of sepsis. Shedding of the endothelial glycocalyx is regarded as a central pathophysiological mechanism causing microvascular dysfunction, contributing to multiple organ failure and death in sepsis. The objective of this study was to investigate whether endothelial glycocalyx thickness at an early stage in septic patients relates to clinical outcome. We measured the perfused boundary region (PBR), which is inversely proportional to glycocalyx thickness, of sublingual microvessels (5–25 µm) using sidestream dark field imaging. The PBR in 21 patients with sepsis was measured within 24 h of admission to the intensive care unit (ICU). In addition, we determined plasma markers of microcirculatory dysfunction and studied their correlation with PBR and mortality. Endothelial glycocalyx thickness in sepsis was significantly lower for non-survivors as compared with survivors, indicated by a higher PBR of 1.97 [1.85, 2.19]µm compared with 1.76 [1.59, 1.97] µm, P=0.03. Admission PBR was associated with hospital mortality with an area under the curve of 0.778 based on the receiver operating characteristic curve. Furthermore, PBR correlated positively with angiopoietin-2 (rho=0.532, P=0.03), indicative of impaired barrier function. PBR did not correlate with Acute Physiology and Chronic Health Evaluation IV (APACHE IV), Sequential Organ Failure Assessment score (SOFA score), lactate, syndecan-1, angiopoietin-1 or heparin-binding protein. An increased PBR within the first 24 h after ICU admission is associated with mortality in sepsis. Further research should be aimed at the pathophysiological importance of glycocalyx shedding in the development of multi-organ failure and at therapies attempting to preserve glycocalyx integrity.

P4991Effects of the glucagon like peptide-1 receptor analogue, sodium-glucose co-transporter 2 and their combination on myocardial work index and vascular function in diabetes after 3-month treatment

Glucagon like peptide-1 analogues (GLP-1A) and sodium-glucose co-transporter 2 (SGLT2) are used in the treatment of type 2 diabetes mellitus (T2DM) We investigated whether the effects of treatment with GLP-1A and SGLT2 and their combination on vascular and cardiac function. Methods A hundred twenty, patients with type 2 diabetes were randomized to receive the insulin +/− metformin (n=30), GLP-1A, liraglutide (n=30), the SGLT-2, empagliflozin (n=30) or their combination (GLP-1R+SGLT-2) for 3 months (n=30). We measured at baseline and after treatment a) pulse wave velocity (PWV), central systolic blood pressure (cSBP) b) the perfusion boundary region (PBR-micrometers) of the sublingual arterial microvessels, as a marker of endothelial glycocalyx thickness. c) global LV longitundinal strain (GLS), peak LV untwisting velocity and global myocardial work index (GWI) derived by pressure–myocardial strain loops using speckle tracking imaging and PWV to GLS ratio, as a marker of ventricular-arterial coupling Results All treatment groups had similar vascular and cardiac markers, glucose levels and HbA1 at inclusion (p>0.05). After treatment, all patients had improved GLS, PWV and increased LV twisting-untwisting velocities (p<0.05). Patients under GLP-1R, SGLT-2 and their combination achieved a significant reduction of PBR, PWV, central SBP and a greater increase of GWI (30%, 25% and 50% respectively) and LV untwisting velocities (p<0.05) than those under insulin (15% reduction of GWI), despite a similar reduction of HbA1 (p<0.05). The combination of GLP-1A and SGLT-2 showed a greater improvement of the measured markers than each treatment alone (table, p<0.05). The reduction of PBR (indicating improvement of glycocalyx thickness) was related with the changes in central SBP (r=0.40) and PWV (r=0.35) (p<0.05). N=120 Insulin GLP-1A SGLT-2 GLP-1A + SGLT-2 P 3-months N=30 N=30 N=30 N=30 PBR, μm 2.27±0.3 2.17±0.3 2.17±0.3 1.99±0.2 <0.05 Central SBP, mmHg 136±20 127±16 126±11 124±10 <0.05 PWV, m/sec 11.7±0.5 9.5±0.5 9.3±0.5 9.2±0.5 <0.05 HbA1 7.1±1.5 6.6±0.3 6.7±0.6 6.2±0.6 0.8 Myocardial work index (GWI), mmHg% 1343±394 1579±245 1364±431 1784±593 <0.05 LV untwisting velocity, deg/sec −116±36 −123±36 −122±36 −130±36 <0.05 PWV/GLS ratio −0.77±0.2 −0.57±0.2 −0.66±0.3 −0.55±0.3 <0.05 Conclusion Three-month treatment with GLP-1A, SGLT-2 and their combination showed a greater improvement of vascular markers, ventricular-arterial coupling and effective cardiac work than insulin treatment in type 2 diabetes.

Effects of high-intensity interval training on microvascular glycocalyx and associated microRNAs

High-intensity interval training (HIIT) has been proposed to exert vasculoprotective effects. This study aimed to evaluate whether HIIT affects the microvasculature, including the endothelial glycocalyx barrier, and to identify associated microRNAs (miRNAs). Fifty healthy participants (23.1 ± 3.0 yr) performed a 4-wk 4 × 30-s all-out running HIIT. Sidestream dark-field imaging was performed at baseline and follow-up to detect changes of the sublingual microvasculature including the endothelial glycocalyx. Exercise parameters were determined by continuous running field test and documentation of high-intensity runs. miRNAs potentially associated with glycocalyx thickness were selected by structured literature search and blood samples for miRNA, and lactate measurements were drawn at baseline and follow-up HIIT. At baseline, a correlation between maximal exercise performance capacity and glycocalyx thickness (determined by perfused boundary region) was detected ( P = 0.045, r = 0.303). Increased exercise performance at follow-up also correlated with glycocalyx thickness ( P = 0.031, r = 0.416), and increased high-intensity sprinting speed was associated with an increased number of perfused vessels ( P = 0.0129, r = 0.449). Literature search identified miR-143, -96-5p, and -24, which were upregulated by HIIT already at baseline and showed an association with peak blood lactate levels after sprints (all P < 0.05). Moreover, increased baseline miR-143 levels predicted increased glycocalyx thickness at follow-up (AUCmiR-143 = 0.92, 95% confidence interval, 0.81–1.0, P = 0.0008). Elevated resting miR-126 levels after the intervention were associated with cell-free versican mRNA levels. We conclude that HIIT induces changes in the endothelial glycocalyx of the microvasculature. Associated miRNAs such as miR-143 may represent a tool for monitoring early vasculoprotective adaptations to physical activity. NEW & NOTEWORTHY High-intensity interval training is known to improve health-related fitness in general and in lifestyle-induced chronic diseases. To visualize microvasculature structure and to detect exercise-induced changes, sublingual sidestream dark-field imaging microscopy was used, and circulating miRNAs were measured. This study shows that exercise-induced changes correlate with associated circulating miRNA, which might be useful for monitoring vasculoprotective effects. Furthermore, sidestream dark-field imaging may represent a sensitive tool for the early detection of exercise-induced systemic vascular changes.

Advanced age results in a diminished endothelial glycocalyx

Age-related microvascular dysfunction is well characterized in rodents and humans, but little is known about the properties of the microvascular endothelial glycocalyx in advanced age. We examined the glycocalyx in microvessels of young and old male C57BL6 mice (young: 6.1 ± 0.1 mo vs. old: 24.6 ± 0.2 mo) using intravital microscopy and transmission electron microscopy and in human participants (young: 29 ± 1 yr vs. old: 60 ± 2 yr) using intravital microscopy. Glycocalyx thickness in mesenteric and skeletal muscle microvessels was 51–54% lower in old compared with young mice. We also observed 33% lower glycocalyx thickness in the sublingual microcirculation of humans in advanced age. The perfused boundary region, a marker of glycocalyx barrier function, was also obtained using an automated capture and analysis system. In advanced age, we observed a 10–22% greater perfused boundary region in mice and humans, indicating a more penetrable glycocalyx. Finally, using this automated analysis system, we examined perfused microvascular density and red blood cell (RBC) fraction. Perfused microvascular density is a marker of microvascular function that reflects the length of perfused microvessel segments in a given area; RBC fraction represents the heterogeneity in RBC presence between microvessel segments. Compared with young, the perfused microvascular density was 16–21% lower and RBC fraction was 5–14% lower in older mice and in older humans. These data provide novel evidence that, across mammalian species, a diminished glycocalyx is present in advanced age and is accompanied by markers of impaired microvascular perfusion. Age-related glycocalyx deterioration may be an important contributor to microvascular dysfunction in older adults and subsequent pathophysiology. NEW & NOTEWORTHY Advanced age is characterized by microvascular dysfunction that contributes to age-related cardiovascular diseases, but little is known about endothelial glycocalyx properties in advanced age. This study reveals, for the first time, lower glycocalyx thickness and barrier function that is accompanied by impaired microvascular perfusion in both mice and humans in advanced age.