Potential Influence of Endothelial Adsorption on the Delayed Time to Maximum Concentration of Biopharmaceuticals.

BACKGROUND AND OBJECTIVES: Maximum plasma concentration of biopharmaceuticals sometimes occurs long after completion of intravenous infusion. The objective of this research was to study the hypothetical adsorption of biopharmaceuticals to endothelium and infusion material, which may theoretically explain this phenomenon. METHODS: Infusion procedures were mimicked in an artificial vessel covered with a confluent monolayer of endothelial cells. Three monoclonal antibodies (MAbs) and C1 inhibitor were studied. RESULTS: Adsorption of MAbs to endothelium was observed followed by release when the vessel was subsequently perfused with buffer. Adsorption to infusion material also occurred to various degrees and in a seemingly random fashion, with a loss of up to 15% during a single flush of the line, but release from the line was not seen. CONCLUSIONS: Our results indicate that adsorption of biopharmaceuticals to endothelium can occur. This observation can explain the increase in plasma concentration after completion of intravenous administration.

Atrasentan Reduces Albuminuria by Restoring the Glomerular Endothelial Glycocalyx Barrier in Diabetic Nephropathy.

Atrasentan, a selective endothelin A receptor antagonist, has been shown to reduce albuminuria in type 2 diabetes. We previously showed that the structural integrity of a glomerular endothelial glycocalyx is required to prevent albuminuria. Therefore we tested the potential of atrasentan to stabilize the endothelial glycocalyx in diabetic apolipoprotein E (apoE)-deficient mice in relation to its antialbuminuric effects. Treatment with atrasentan (7.5 mg/kg/day) for 4 weeks reduced urinary albumin-to-creatinine ratios by 26.0 ± 6.5% (P < 0.01) in apoE knockout (KO) mice with streptozotocin-induced diabetes consuming an atherogenic diet, without changes in gross glomerular morphology, systemic blood pressure, and blood glucose concentration. Endothelial cationic ferritin surface coverage, investigated using large-scale digital transmission electron microscopy, revealed that atrasentan treatment increases glycocalyx coverage in diabetic apoE KO mice from 40.7 ± 3.2% to 81.0 ± 12.5% (P < 0.05). This restoration is accompanied by increased renal nitric oxide concentrations, reduced expression of glomerular heparanase, and a marked shift in the balance of M1 and M2 glomerular macrophages. In vitro experiments with endothelial cells exposed to laminar flow and cocultured with pericytes confirmed that atrasentan reduced endothelial heparanase expression and increased glycocalyx thickness in the presence of a diabetic milieu. Together these data point toward a role for the restoration of endothelial function and tissue homeostasis through the antialbuminuric effects of atrasentan, and they provide a mechanistic explanation for the clinical observations of reduced albuminuria with atrasentan in diabetic nephropathy.

The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and ß-catenin protein expression.

Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and ß-catenin and can induce mRNA translation mediated by the 3'UTR of these genes. Reduced quaking levels attenuated VE-cadherin and ß-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity.

A microscopic view on the renal endothelial glycocalyx.

Endothelial cells perform key homeostatic functions such as regulating blood flow, permeability, and aiding immune surveillance for pathogens. While endothelial activation serves normal physiological adaptation, maladaptation of these endothelial functions has been identified as an important effector mechanism in the progression of renal disease as well as the associated development of cardiovascular disease. The primary interface between blood and the endothelium is the glycocalyx. This carbohydrate-rich gel-like structure with its associated proteins mediates most of the regulatory functions of the endothelium. Because the endothelial glycocalyx is a highly dynamic and fragile structure ex vivo, and traditional tissue processing for staining and perfusion-fixation usually results in a partial or complete loss of the glycocalyx, studying its dimensions and function has proven to be challenging. In this review, we will outline the core functions of the glycocalyx and focus on different techniques to study structure-function relationships in kidney and vasculature.

Deeper penetration of erythrocytes into the endothelial glycocalyx is associated with impaired microvascular perfusion.

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 ß: -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.

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: Spearman's ρ=0.31; P<0.05; perfused boundary region versus angiopoietin-2: Spearman's 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.

Glomerular endothelial surface layer acts as a barrier against albumin filtration.

Glomerular endothelium is highly fenestrated, and its contribution to glomerular barrier function is the subject of debate. In recent years, a polysaccharide-rich endothelial surface layer (ESL) has been postulated to act as a filtration barrier for large molecules, such as albumin. To test this hypothesis, we disturbed the ESL in C57Bl/6 mice using long-term hyaluronidase infusion for 4 weeks and monitored albumin passage using immunolabeling and correlative light-electron microscopy that allows for complete and integral assessment of glomerular albumin passage. ESL ultrastructure was visualized by transmission electron microscopy using cupromeronic blue and by localization of ESL binding lectins using confocal microscopy. We demonstrate that glomerular fenestrae are filled with dense negatively charged polysaccharide structures that are largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact. Both retention of native ferritin [corrected] in the glomerular basement membrane and systemic blood pressure were unaltered. Enzyme treatment, however, induced albumin passage across the endothelium in 90% of glomeruli, whereas this could not be observed in controls. Yet, there was no net albuminuria due to binding and uptake of filtered albumin by the podocytes and parietal epithelium. ESL structure and function completely recovered within 4 weeks on cessation of hyaluronidase infusion. Thus, the polyanionic ESL component, hyaluronan, is a key component of the glomerular endothelial protein permeability barrier.

The endothelial glycocalyx as a potential modifier of the hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (HUS) is a renal disease due to complement dysregulation. Many of the known causes of atypical HUS originate from genetic mutations of complement regulatory proteins, such as complement factor H (CFH) and thrombomodulin. However, atypical HUS has only a genetic penetrance of 40-50% of the cases and usually appears in adulthood. We introduce a novel factor that may be involved in the onset and development of atypical HUS, i.e. the endothelial surface glycocalyx. The glycocalyx is a highly interactive matrix covering the luminal side of vascular endothelial cells and consists of glycosaminoglycans, proteoglycans and glycoproteins, which has an important role in maintaining homeostasis of the vasculature. The surface-bound glycocalyx glycosaminoglycan constituent heparan sulfate is crucial for CFH binding and function, both in recognition of host tissue and prevention of spontaneous complement activation via the alternative pathway. Most of the clinically relevant genetic mutations in CFH result in incorrect binding to heparan sulfate. In addition, a role between proper function of thrombomodulin and the endothelial glycocalyx has also been observed. We suggest that not only changes in binding properties of the complement regulatory proteins play a role but also changes in the endothelial glycocalyx are involved in increased risk of clinical manifestation of atypical HUS. Finally, vascular glycocalyx heterogeneity in turn could dictate the specific vulnerability of the glomerular vascular bed in atypical HUS and may provide new therapeutic targets to intervene with endothelial cell activation and local complement pathway regulation.