Fasting mimicking diet in diabetic mice partially preserves glomerular endothelial glycocalyx coverage, without changing the diabetic metabolic environment.

Intermittent fasting has become of interest for its possible metabolic benefits and reduction of inflammation and oxidative damage, all of which play a role in the pathophysiology of diabetic nephropathy. We tested in a streptozotocin (60 mg/kg)-induced diabetic apolipoprotein E knockout mouse model whether repeated fasting mimicking diet (FMD) prevents glomerular damage. Diabetic mice received 5 FMD cycles in 10 wk, and during cycles 1 and 5 caloric measurements were performed. After 10 wk, glomerular endothelial morphology was determined together with albuminuria, urinary heparanase-1 activity, and spatial mass spectrometry imaging to identify specific glomerular metabolic dysregulation. During FMD cycles, blood glucose levels dropped while a temporal metabolic switch was observed to increase fatty acid oxidation. Overall body weight at the end of the study was reduced together with albuminuria, although urine production was dramatically increased without affecting urinary heparanase-1 activity. Weight loss was found to be due to lean mass and water, not fat mass. Although capillary loop morphology and endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced together with the presence of UDP-glucuronic acid. Mass spectrometry imaging further revealed reduced protein catabolic breakdown products and increased oxidative stress, not different from diabetic mice. In conclusion, although FMD preserves partially glomerular endothelial glycocalyx, loss of lean mass and increased glomerular oxidative stress argue whether such diet regimes are safe in patients with diabetes.NEW & NOTEWORTHY Repeated fasting mimicking diet (FMD) partially prevents glomerular damage in a diabetic mouse model; however, although endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced in the presence of UDP-glucuronic acid. The weight loss observed was of lean mass, not fat mass, and increased glomerular oxidative stress argue whether such a diet is safe in patients with diabetes.

Heparan sulfate mimetic fucoidan restores the endothelial glycocalyx and protects against dysfunction induced by serum of COVID-19 patients in the intensive care unit.

Accumulating evidence proves that endothelial dysfunction is involved in coronavirus disease 2019 (COVID-19) progression. We previously demonstrated that the endothelial surface glycocalyx has a critical role in maintenance of vascular integrity. Here, we hypothesised that serum factors of severe COVID-19 patients affect the glycocalyx and result in endothelial dysfunction. We included blood samples of 32 COVID-19 hospitalised patients at the Leiden University Medical Center, of which 26 were hospitalised in an intensive care unit (ICU) and six on a non-ICU hospital floor; 18 of the samples were obtained from convalescent patients 6 weeks after hospital discharge, and 12 from age-matched healthy donors (control) during the first period of the outbreak. First, we determined endothelial (angiopoietin 2 (ANG2)) and glycocalyx degradation (soluble thrombomodulin (sTM) and syndecan-1 (sSDC1)) markers in plasma. In the plasma of COVID-19 patients, circulating ANG2 and sTM were elevated in patients in the ICU. Primary lung microvascular endothelial cell (HPMEC) and human glomerular microvascular endothelial cell (GEnC) cultured in the presence of these sera led to endothelial cell glycocalyx degradation, barrier disruption, inflammation and increased coagulation on the endothelial surface, significantly different compared to healthy control and non-ICU patient sera. These changes could all be restored in the presence of fucoidan. In conclusion, our data highlight the link between endothelial glycocalyx degradation, barrier failure and induction of a procoagulant surface in COVID-19 patients in ICU which could be targeted earlier in disease by the presence of heparan sulfate mimetics.

Netrin-4 expression by human endothelial cells inhibits endothelial inflammation and senescence.

Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured with electric cell-substrate impedance sensing, as well as in an 'organ-on-a-chip' microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated ß-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health.

Loss of Endothelial Glycocalyx Hyaluronan Impairs Endothelial Stability and Adaptive Vascular Remodeling After Arterial Ischemia.

We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia.

Lowering the increased intracellular pH of human-induced pluripotent stem cell-derived endothelial cells induces formation of mature Weibel-Palade bodies.

Differentiation of human-induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial-like cells (hiPSC-derived endothelial cells [hiPSC-ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC-ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel-Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC-ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC-ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC-ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC-ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC-EC. Mechanistically, pro-VWF dimers require the lower pH environment of the trans-Golgi network for maturation and tubulation. These data show that while hiPSC-ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function.

Endothelial Semaphorin 3F Maintains Endothelial Barrier Function and Inhibits Monocyte Migration.

In normal physiology, endothelial cells (ECs) form a vital barrier between the blood and underlying tissue controlling leukocyte diapedesis and vascular inflammation. Emerging data suggest that neuronal guidance cues, typically expressed during development, have roles outside the nervous system in vascular biology and immune responses. In particular, Class III semaphorins have been reported to affect EC migration and angiogenesis. While ECs express high levels of semaphorin 3F (SEMA3F), little is known about its function in mature ECs. Here we show that SEMA3F expression is reduced by inflammatory stimuli and increased by laminar flow. Endothelial cells exposed to laminar flow secrete SEMA3F, which subsequently binds to heparan sulfates on the surface of ECs. However, under pro-inflammatory conditions, reduced levels of SEMA3F make ECs more prone to monocyte diapedesis and display impaired barrier function as measured with an electric cell-substrate impedance sensing system and a microfluidic system. In addition, we demonstrate that SEMA3F can directly inhibit the migration of activated monocytes. Taken together, our data suggest an important homeostatic function for EC-expressed SEMA3F, serving as a mediator of endothelial quiescence.

Shear Stress Regulation of Endothelial Glycocalyx Structure Is Determined by Glucobiosynthesis.

OBJECTIVE: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with 13C-labeled glucose. CONCLUSIONS: These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.

Closing the Mitochondrial Permeability Transition Pore in hiPSC-Derived Endothelial Cells Induces Glycocalyx Formation and Functional Maturation.

Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality.

Glomerular Function and Structural Integrity Depend on Hyaluronan Synthesis by Glomerular Endothelium.

BACKGROUND: A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function. METHODS: To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy. RESULTS: Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2. CONCLUSIONS: Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrix that is required for glomerular structure and function and lost in diabetic nephropathy.

Vascular bioengineering of scaffolds derived from human discarded transplant kidneys using human pluripotent stem cell-derived endothelium.

The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re-endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re-endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell -derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re-endothelialized scaffold could, in contrast to the non-re-endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney.

Systemic Monocyte Chemotactic Protein-1 Inhibition Modifies Renal Macrophages and Restores Glomerular Endothelial Glycocalyx and Barrier Function in Diabetic Nephropathy.

Inhibition of monocyte chemotactic protein-1 (MCP-1) with the Spiegelmer emapticap pegol (NOX-E36) shows long-lasting albuminuria-reducing effects in diabetic nephropathy. MCP-1 regulates inflammatory cell recruitment and differentiation of macrophages. Because the endothelial glycocalyx is also reduced in diabetic nephropathy, we hypothesized that MCP-1 inhibition restores glomerular barrier function through influencing macrophage cathepsin L secretion, thus reducing activation of the glycocalyx-degrading enzyme heparanase. Four weeks of treatment of diabetic Apoe knockout mice with the mouse-specific NOX-E36 attenuated albuminuria without any change in systemic hemodynamics, despite persistent loss of podocyte function. MCP-1 inhibition, however, increased glomerular endothelial glycocalyx coverage, with preservation of heparan sulfate. Mechanistically, both glomerular cathepsin L and heparanase expression were reduced. MCP-1 inhibition resulted in reduced CCR2-expressing Ly6Chi monocytes in the peripheral blood, without affecting overall number of kidney macrophages at the tissue level. However, the CD206+/Mac3+ cell ratio, as an index of presence of anti-inflammatory macrophages, increased in diabetic mice after treatment. Functional analysis of isolated renal macrophages showed increased release of IL-10, whereas tumor necrosis factor and cathepsin L release was reduced, further confirming polarization of tissue macrophages toward an anti-inflammatory phenotype during mouse-specific NOX-E36 treatment. We show that MCP-1 inhibition restores glomerular endothelial glycocalyx and barrier function and reduces tissue inflammation in the presence of ongoing diabetic injury, suggesting a therapeutic potential for NOX-E36 in diabetic nephropathy.