Tissue-dependent variation in NO free radical induction.

<p>(A) EPR spectrum of frozen kidney samples. The characteristic triplet structure of the mononitrosyl-iron complex (MNIC, double-headed arrow) centers around g  =  2.035 and represents the formation of local nitric oxide in 334–370 mg tissue. (B-D) Quantification of nitric oxide formation in kidney, liver and heart tissue, shown as mean pmol MNIC / mg wet tissue ± SD, n = 7–9. E) Plasma ADMA concentrations, shows as mean ± SD, n = 8. *P<0.05, compared with ApoE; #P<0.05 compared with DM. ApoE = ApoE KO mice, DM = diabetic apoE KO mice, DM + S = diabetic apoE KO mice + sepiapterin.</p

Increased NO levels affect endothelial glycocalyx non-uniformly.

<p>(A) Representative microscopic images of cationic ferritin (TEM; top), heparanase (HPSE, immunofluorescence; middle) and cathepsin L (CTSL; bottom) in glomeruli of apoE KO mice (apoE), diabetic apoE KO mice (DM) and diabetic apoE KO mice treated with sepiapterin (DM + S). (B) Quantification of endothelial cationic ferritin coverage in 6–8 capillary loops in 9 glomeruli of 3 mice, shown as mean percentage of total capillary length ± SD, (C) Quantification of glomerular heparanase expression, shown as mean area percentage ± SD. (D) Quantification of glomerular cathepsin L expression, shown as mean area percentage ± SD. *P<0.05 compared with ApoE, n = 6–8. Scale bars: 500 nm in TEM images; 20 μm in fluorescent and light microscopic images.</p

Sepiapterin does not reduce albuminuria in diabetic apoE KO mice.

<p>(A) PAS-stained glomeruli of apoE KO mice (apoE), diabetic apoE KO mice (DM) and diabetic apoE KO mice treated with sepiapterin (DM + S), showing heterogeneous diabetic lesions 14 weeks after induction of diabetes with STZ (20). Scale bars: 20 μm. Sepiapterin did not affect mesangial area (B,C), nor blood glucose concentrations (D). Data are shown as mean ± SD, *P<0.05 compared with apoE, n = 8. (E) Albumin-creatinine ratios (ACR) at baseline, 2- and 4 weeks after treatment, as indicated by mean ± SEM, *P<0.05 compared with apoE, n = 14–23.</p

Experimental set-up for assessment of NO bioavailability.

<p>(A) male ApoE KO mice (<i>B6</i>.<i>129P2- Apoe</i>^{<i>tm1Unc</i>}<i>/J)</i> were injected with citrate buffer ± STZ. Diabetic mice received cholesterol enriched diet and insulin from week 8 onwards. At 18 week of age, diabetic mice were treated with sepiapterin or received normal drinking water for 4 weeks. Urine was collected upon commencing with the experimental procedure and after 2 and 4 weeks of treatment. At 22 weeks, plasma was collected and the mice were sacrificed.</p

Schematic illustration on relation between glycocalyx accessibility and microvascular perfusion regulation.

<p><b>A</b>) Healthy state: Intact glycocalyx prevents red blood cells (RBC, red dots) from penetrating into its domain, reflected by a low perfused boundary region (PBR), and nicely aligned elongated RBC. The vessels are well perfused (higher tube hematocrit of microvessel and elongated shape of erythrocyte) resulting in a higher percentage of vessel segments with RBC present at any particular time point (high RBC filling percentage). <b>B</b>) Risk State: Altered composition of glycocalyx (lined dots) allows RBCs to penetrate deeper into the glycocalyx, closer to the anatomical border of lumen (endothelium), reflected by the high PBR. Due to the widening of RBC distribution width and volume, there is more space in between each RBC, as shown by decreased RBC filling percentage (less positive contrast per vascular segment per time point). Also, prolonged state of glycocalyx degradation leads to edematous and non-functioning vessels, leading to shorter vessel density per area of tissue (reduced valid microvascular density in risk PBR), depicted by the loss of bottom vessel.</p

Sidestream darkfield imaging derived variables characteristics.

<p>Data is presented as mean ± standard deviation.</p

Linear regression analysis show association between perfused boundary region and microvascular perfusion parameters.

<p>*Dependent variable: Perfused boundary region (PBR).</p>†<p>95% confidence interval for regression coefficient β.</p>‡<p>Linear regression analysis adjusted for age, sex and BMI.</p>§<p>Valid microvascular density expressed as millimeter of microvessel length per mm² of area of tissue (mm/mm²) for linear regression analysis due to difference in scale from PBR.</p

Glycocheck algorithm on endothelial PBR determination and microvascular perfusion properties.

<p><b>A</b>) Red blood cells (RBC) are detected through reflection of light emitting diodes by hemoglobin. Images captured by the sidestream darkfield camera are sent to the computer for quality checks and assessment. The black contrast is the perfused lumen of the vessels. <b>B</b>) In each recording, the software automatically places the vascular segments (green), every 10 µm along the vascular segments (black contrast). <b>C</b>) After the acquisition, for the analysis, the software undergoes several quality check in the first frame of each recording (see text), to select vascular segments with sufficient quality for further analysis. Invalid vascular segments (yellow) are distinguished from the valid vascular segments (green). During the whole recording session of 40 frames, the percentage of time in which a particular valid vascular segment has RBCs present is used to calculate RBC filling percentage. <b>D</b>) Depiction of the concept of glycocalyx thickness by lateral RBC movement is shown here. <b>E</b>) For each vascular segment, the intensity profile is calculated to derive median RBC column width. <b>F</b>) Then, the distribution of RBC column width is used to calculate the perfused diameter, median RBC column width, and subsequently the perfused boundary region (PBR).</p

Scatterplot between PBR and outcomes of microvascular perfusion.

<p>The perfused boundary region (PBR), a measurement for glycocalyx accessibility to red blood cells (RBC), is associated significantly with spatio-temporal aspects of microvascular perfusion variables: <b>A</b>) RBC filling percentage (percentage of time in which a particular vascular segment is perfused) <b>B</b>) Valid microvascular density. In particular, lower PBR (less accessible glycocalyx, thus a better and thicker glycocalyx) is associated with higher RBC filling percentage (temporal perfusion).</p

General Clinical Characteristics.

<p>Hb, hemoglobin; Hct, hematocrit; BP, blood pressure. Data is presented as mean ± standard deviation.</p