Role of dietary interventions on microvascular health in South-Asian Surinamese people with type 2 diabetes in the Netherlands: A randomized controlled trial.

BACKGROUND/OBJECTIVES: We investigated whether dietary interventions, i.e. a fasting mimicking diet (FMD, Prolon®) or glycocalyx mimetic supplementation (EndocalyxTM) could stabilize microvascular function in Surinamese South-Asian patients with type 2 diabetes (SA-T2DM) in the Netherlands, a patient population more prone to develop vascular complications. SUBJECTS/METHODS: A randomized, placebo controlled, 3-arm intervention study was conducted in 56 SA-T2DM patients between 18 and 75 years old, for 3 consecutive months, with one additional follow up measurement 3 months after the last intervention. Sublingual microcirculation was assessed with SDF-imaging coupled to the GlycoCheckTM software, detecting red blood cell velocity, capillary density, static and dynamic perfused boundary region (PBR), and the overall microvascular health score (MVHS). Linear mixed models and interaction analysis were used to investigate the effects the interventions had on microvascular function. RESULTS: Despite a temporal improvement in BMI and HbA1c after FMD the major treatment effect on microvascular health was worsening for RBC-velocity independent PBRdynamic, especially at follow-up. Glycocalyx supplementation, however, reduced urinary MCP-1 presence and improved both PBRdynamic and MVHSdynamic, which persisted at follow-up. CONCLUSIONS: We showed that despite temporal beneficial changes in BMI and HbA1c after FMD, this intervention is not able to preserve microvascular endothelial health in Dutch South-Asian patients with T2DM. In contrast, glycocalyx mimetics preserves the microvascular endothelial health and reduces the inflammatory cytokine MCP-1. CLINICAL STUDY REGISTRATION: NCT03889236.

Ethnic Disparities in Lipid Metabolism and Clinical Outcomes between Dutch South Asians and Dutch White Caucasians with Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) poses a higher risk for complications in South Asian individuals compared to other ethnic groups. To shed light on potential mediating factors, we investigated lipidomic changes in plasma of Dutch South Asians (DSA) and Dutch white Caucasians (DwC) with and without T2DM and explore their associations with clinical features. Using a targeted quantitative lipidomics platform, monitoring over 1000 lipids across 17 classes, along with 1H NMR based lipoprotein analysis, we studied 51 healthy participants (21 DSA, 30 DwC) and 92 T2DM patients (47 DSA, 45 DwC) from the MAGNetic resonance Assessment of VICTOza efficacy in the Regression of cardiovascular dysfunction in type 2 dIAbetes mellitus (MAGNA VICTORIA) study. This comprehensive mapping of the circulating lipidome allowed us to identify relevant lipid modules through unbiased weighted correlation network analysis, as well as disease and ethnicity related key mediatory lipids. Significant differences in lipidomic profiles, encompassing various lipid classes and species, were observed between T2DM patients and healthy controls in both the DSA and DwC populations. Our analyses revealed that healthy DSA, but not DwC, controls already exhibited a lipid profile prone to develop T2DM. Particularly, in DSA-T2DM patients, specific lipid changes correlated with clinical features, particularly diacylglycerols (DGs), showing significant associations with glycemic control and renal function. Our findings highlight an ethnic distinction in lipid modules influencing clinical outcomes in renal health. We discover distinctive ethnic disparities of the circulating lipidome and identify ethnicity-specific lipid markers. Jointly, our discoveries show great potential as personalized biomarkers for the assessment of glycemic control and renal function in DSA-T2DM individuals.

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.

Sodium Butyrate as Key Regulator of Mitochondrial Function and Barrier Integrity of Human Glomerular Endothelial Cells.

The gut microbiota has emerged as an important modulator of cardiovascular and renal homeostasis. The composition of gut microbiota in patients suffering from chronic kidney disease (CKD) is altered, where a lower number of bacteria producing short chain fatty acids (SCFAs) is observed. It is known that SCFAs, such as butyrate and acetate, have protective effects against cardiovascular diseases and CKD but their mechanisms of action remain largely unexplored. In the present study, we investigated the effect of butyrate and acetate on glomerular endothelial cells. Human glomerular microvascular endothelial cells (hgMVECs) were cultured and exposed to butyrate and acetate and their effects on cellular proliferation, mitochondrial mass and metabolism, as well as monolayer integrity were studied. While acetate did not show any effects on hgMVECs, our results revealed that butyrate reduces the proliferation of hgMVECs, strengthens the endothelial barrier through increased expression of VE-cadherin and Claudin-5 and promotes mitochondrial biogenesis. Moreover, butyrate reduces the increase in oxygen consumption induced by lipopolysaccharides (LPS), revealing a protective effect of butyrate against the detrimental effects of LPS. Taken together, our data show that butyrate is a key player in endothelial integrity and metabolic homeostasis.

Sex-specific association between microvascular health and coagulation parameters: the Netherlands Epidemiology of Obesity study.

BACKGROUND: Microvascular dysfunction is a growing determinant of sex differences in coronary heart disease (CHD). Dysregulation of the coagulation system is involved in CHD pathogenesis and can be induced by endothelial glycocalyx (EG) perturbation. However, little is known about the link between EG function and coagulation parameters in population-based studies on sex specificity. OBJECTIVES: We sought to examine the sex differences in the relationship between EG function and coagulation parameters in a middle-aged Dutch population. METHODS: Using baseline measurements of 771 participants from the Netherlands Epidemiology of Obesity study (age, 56 years [IQR, 51-61 years]; 53% women; body mass index, 27.9 kg/m2 [IQR, 25.1-30.9 kg/m2]), associations between glycocalyx-related perfused boundary region (PBR) derived using sidestream dark-field imaging and coagulation parameters (factor [F]VIII/IX/XI; thrombin generation parameters; and fibrinogen) were investigated using linear regression analyses, adjusting for possible confounders (including C-reactive protein, leptin, and glycoprotein acetyls), followed by sex-stratified analyses. RESULTS: There was a sex difference in the associations between PBR and coagulation parameters. Particularly in women, 1-SD PBR (both total and feed vessel, indicating poorer glycocalyx status) was associated with higher FIX activity ([1.8%; 95% CI, 0.3%-3.3%] and [2.0%; 95% CI, 0.5%-3.4%], respectively) and plasma fibrinogen levels ([5.1 mg/dL; 95% CI, 0.4-9.9 mg/dL] and [5.8 mg/dL; 95% CI, 1.1-10.6 mg/dL], respectively). Furthermore, 1-SD PBRcapillary was associated with higher FVIII activity (3.5%; 95% CI, 0.4%-6.5%) and plasma fibrinogen levels (5.3 mg/dL; 95% CI, 0.6-10.0 mg/dL). CONCLUSION: We revealed a sex-specific association between microcirculatory health and procoagulant status, which suggests that microvascular health be considered during early development of CHD in women.

Altered high-density lipoprotein composition is associated with risk for complications in type 2 diabetes mellitus in South Asian descendants: A cross-sectional, case-control study on lipoprotein subclass profiling.

BACKGROUND: Composition of high-density lipoproteins (HDL) is emerging as an important determinant in the development of microvascular complications in type 2 diabetes mellitus (T2DM). Dutch South Asian (DSA) individuals with T2DM display an increased risk of microvascular complications compared with Dutch white Caucasian (DwC) individuals with T2DM. In this study, we aimed to investigate whether changes in HDL composition associate with increased microvascular risk in this ethnic group and lead to new lipoprotein biomarkers. MATERIALS AND METHODS: Using 1 H nuclear magnetic resonance spectroscopy and Bruker IVDr Lipoprotein Subclass Analysis (B.I.LISA) software, plasma lipoprotein changes were determined in 51 healthy individuals (30 DwC, 21 DSA) and 92 individuals with T2DM (45 DwC, 47 DSA) in a cross-sectional, case-control study. Differential HDL subfractions were investigated using multinomial logistic regression analyses, adjusting for possible confounders including BMI and diabetes duration. RESULTS: We identified HDL compositional differences between healthy and diabetic individuals in both ethnic groups. Specifically, levels of apolipoprotein A2 and HDL-4 subfractions were lower in DSA compared with DwC with T2DM. Apolipoprotein A2 and HDL-4 subfractions also negatively correlated with waist circumference, waist-to-hip ratio, haemoglobin A1c, glucose levels and disease duration in DSA with T2DM, and associated with increased incidence of microvascular complications. CONCLUSION: While HDL composition differed between controls and T2DM in both ethnic groups, the lower levels of lipid content in the smallest HDL subclass (HDL-4) in DSA with T2DM appeared to be more clinically relevant, with higher odds of having diabetes-related pan-microvascular complications such as retinopathy and neuropathy. These typical differences in HDL could be used as ethnicity-specific T2DM biomarkers.

Phosphatidylinositol metabolism of the renal proximal tubule S3 segment is disturbed in response to diabetes.

Diabetes is a main risk factor for kidney disease, causing diabetic nephropathy in close to half of all patients with diabetes. Metabolism has recently been identified to be decisive in cell fate decisions and repair. Here we used mass spectrometry imaging (MSI) to identify tissue specific metabolic dysregulation, in order to better understand early diabetes-induced metabolic changes of renal cell types. In our experimental diabetes mouse model, early glomerular glycocalyx barrier loss and systemic metabolic changes were observed. In addition, MSI targeted at small molecule metabolites and glycero(phospho)lipids exposed distinct changes upon diabetes in downstream nephron segments. Interestingly, the outer stripe of the outer medullar proximal tubular segment (PT_S3) demonstrated the most distinct response compared to other segments. Furthermore, phosphatidylinositol lipid metabolism was altered specifically in PT_S3, with one of the phosphatidylinositol fatty acid tails being exchanged from longer unsaturated fatty acids to shorter, more saturated fatty acids. In acute kidney injury, the PT_S3 segment and its metabolism are already recognized as important factors in kidney repair processes. The current study exposes early diabetes-induced changes in membrane lipid composition in this PT_S3 segment as a hitherto unrecognized culprit in the early renal response to diabetes.

Ethnic differences in urinary monocyte chemoattractant protein-1 and heparanase-1 levels in individuals with type 2 diabetes: the HELIUS study.

INTRODUCTION: We aimed to investigate ethnic differences in two urinary inflammatory markers in participants with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: We included 55 Dutch, 127 South-Asian Surinamese, 92 African Surinamese, 62 Ghanaian, 74 Turkish and 88 Moroccan origin participants with T2DM from the HEalthy LIfe in an Urban Setting study. Using linear regression analyses, we investigated differences in urinary monocyte chemoattractant protein-1 (MCP-1) and heparanase-1 (HPSE-1) levels across ethnic minorities compared with Dutch. Associations between the urinary markers and albuminuria (albumin:creatinine ratio (ACR)) was investigated per ethnicity. RESULTS: Urinary MCP-1 levels were higher in the Moroccan participants (0.15 log ng/mmol, 95% CI 0.05 to 0.26) compared with Dutch after multiple adjustments. Urinary HPSE-1 levels were lower in the African Surinamese and Ghanaian participants compared with the Dutch, with a difference of -0.16 log mU/mmol (95% CI -0.29 to -0.02) in African Surinamese and -0.16 log mU/mmol (95% CI -0.31 to -0.00) in Ghanaian after multiple adjustments. In all ethnic groups except the Dutch and Ghanaian participants, MCP-1 was associated with ACR. This association remained strongest after multiple adjustment in South-Asian and African Surinamese participants, with an increase in log ACR of 1.03% (95% CI 0.58 to 1.47) and 1.23% (95% CI 0.52 to 1.94) if log MCP-1 increased 1%. Only in the Dutch participants, an association between HPSE-1 and ACR was found, with increase in log ACR of 0.40% (95% CI 0.04 to 0.76) if log HPSE-1 increased 1%. CONCLUSIONS: We found ethnic differences in urinary MCP-1 and HPSE-1 levels, in a multi-ethnic cohort of participants with T2DM. In addition, we found ethnic differences in the association of MCP-1 and HPSE-1 levels with albuminuria. These findings suggest differences in renal inflammation across ethnic groups.

Spatial dynamic metabolomics identifies metabolic cell fate trajectories in human kidney differentiation.

Accumulating evidence demonstrates important roles for metabolism in cell fate determination. However, it is a challenge to assess metabolism at a spatial resolution that acknowledges both heterogeneity and cellular dynamics in its tissue microenvironment. Using a multi-omics platform to study cell-type-specific dynamics in metabolism in complex tissues, we describe the metabolic trajectories during nephrogenesis in the developing human kidney. Exploiting in situ analysis of isotopic labeling, a shift from glycolysis toward fatty acid ß-oxidation was observed during the differentiation from the renal vesicle toward the S-shaped body and the proximal tubules. In addition, we show that hiPSC-derived kidney organoids are characterized by a metabolic immature phenotype that fails to use mitochondrial long-chain fatty acids for energy metabolism. Furthermore, supplementation of butyrate enhances tubular epithelial differentiation and maturation in cultured kidney organoids. Our findings highlight the relevance of understanding metabolic trajectories to efficiently guide stem cell differentiation.

Analyzing cell-type-specific dynamics of metabolism in kidney repair.

A common drawback of metabolic analyses of complex biological samples is the inability to consider cell-to-cell heterogeneity in the context of an organ or tissue. To overcome this limitation, we present an advanced high-spatial-resolution metabolomics approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) combined with isotope tracing. This method allows mapping of cell-type-specific dynamic changes in central carbon metabolism in the context of a complex heterogeneous tissue architecture, such as the kidney. Combined with multiplexed immunofluorescence staining, this method can detect metabolic changes and nutrient partitioning in targeted cell types, as demonstrated in a bilateral renal ischemia-reperfusion injury (bIRI) experimental model. Our approach enables us to identify region-specific metabolic perturbations associated with the lesion and throughout recovery, including unexpected metabolic anomalies in cells with an apparently normal phenotype in the recovery phase. These findings may be relevant to an understanding of the homeostatic capacity of the kidney microenvironment. In sum, this method allows us to achieve resolution at the single-cell level in situ and hence to interpret cell-type-specific metabolic dynamics in the context of structure and metabolism of neighboring cells.

Vasculogenesis in kidney organoids upon transplantation.

Human induced pluripotent stem cell-derived kidney organoids have potential for disease modeling and to be developed into clinically transplantable auxiliary tissue. However, they lack a functional vasculature, and the sparse endogenous endothelial cells (ECs) are lost upon prolonged culture in vitro, limiting maturation and applicability. Here, we use intracoelomic transplantation in chicken embryos followed by single-cell RNA sequencing and advanced imaging platforms to induce and study vasculogenesis in kidney organoids. We show expansion of human organoid-derived ECs that reorganize into perfused capillaries and form a chimeric vascular network with host-derived blood vessels. Ligand-receptor analysis infers extensive potential interactions of human ECs with perivascular cells upon transplantation, enabling vessel wall stabilization. Perfused glomeruli display maturation and morphogenesis to capillary loop stage. Our findings demonstrate the beneficial effect of vascularization on not only epithelial cell types, but also the mesenchymal compartment, inducing the expansion of ´on target´ perivascular stromal cells, which in turn are required for further maturation and stabilization of the neo-vasculature. The here described vasculogenic capacity of kidney organoids will have to be deployed to achieve meaningful glomerular maturation and kidney morphogenesis in vitro.

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.

Mutations in the heparan sulfate backbone elongating enzymes EXT1 and EXT2 have no major effect on endothelial glycocalyx and the glomerular filtration barrier.

In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology.

Circulating angiopoietin-2 and angiogenic microRNAs associate with cerebral small vessel disease and cognitive decline in older patients reaching end-stage renal disease.

BACKGROUND: The prevalence of end-stage renal disease (ESRD) is increasing worldwide, with the majority of new ESRD cases diagnosed in patients >60 years of age. These older patients are at increased risk for impaired cognitive functioning, potentially through cerebral small vessel disease (SVD). Novel markers of vascular integrity may be of clinical value for identifying patients at high risk for cognitive impairment. METHODS: We aimed to associate the levels of angiopoietin-2 (Ang-2), asymmetric dimethylarginine and a selection of eight circulating angiogenic microRNAs (miRNAs) with SVD and cognitive impairment in older patients reaching ESRD that did not yet initiate renal replacement therapy (n = 129; mean age 75.3 years, mean eGFR 16.4 mL/min). We assessed brain magnetic resonance imaging changes of SVD (white matter hyperintensity volume, microbleeds and the presence of lacunes) and measures of cognition in domains of memory, psychomotor speed and executive function in a neuropsychological test battery. RESULTS: Older patients reaching ESRD showed an unfavourable angiogenic profile, as indicated by aberrant levels of Ang-2 and five angiogenic miRNAs (miR-27a, miR-126, miR-132, miR-223 and miR-326), compared with healthy persons and patients with diabetic nephropathy. Moreover, Ang-2 was associated with SVD and with the domains of psychomotor speed and executive function, while miR-223 and miR-29a were associated with memory function. CONCLUSIONS: Taken together, these novel angiogenic markers might serve to identify older patients with ESRD at risk of cognitive decline, as well as provide insights into the underlying (vascular) pathophysiology.

Microvascular differences in individuals with obesity at risk of developing cardiovascular disease.

OBJECTIVE: This study aimed to investigate microvascular differences in individuals with obesity at risk for developing cardiovascular disease. METHODS: In this cross-sectional Netherlands Epidemiology of Obesity study, participant sublingual microcirculation was assessed with a newly developed GlycoCheck software (Microvascular Health Solutions Inc., Salt Lake City, Utah), which integrates red blood cell velocity within the smallest capillaries (4-7 µm) and feed vessels (>10 µm). Framingham Risk Score was used to calculate 10-year cardiovascular risk, divided into low-, intermediate-, and high-risk groups. ANOVA was used to evaluate microvascular differences among the groups. RESULTS: A total of 813 participants were included. The high-risk group (n = 168) was characterized by differences in the microvasculature compared with the low-risk group (n = 392): the high-risk group had a 49% reduction in the number of smallest capillaries and a 9.1-µm/s (95% CI: 5.2-12.9) higher red blood cell velocity in the feed vessels. No differences in velocity-corrected perfused boundary regions were found. CONCLUSIONS: It was observed that, with adding red blood cell velocity to the software, sidestream dark field imaging is able to detect microcirculatory differences in a cohort of individuals with obesity at risk for developing cardiovascular disease.

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.

Reduced Glomerular Endothelial Thrombomodulin Is Associated with Glomerular Macrophage Infiltration in Diabetic Nephropathy.

The endothelial glycoprotein thrombomodulin regulates coagulation, inflammation, and apoptosis. In diabetic mice, reduced thrombomodulin function results in diabetic nephropathy (DN). Furthermore, thrombomodulin treatment reduces renal inflammation and fibrosis. Herein, thrombomodulin expression was examined in human kidney samples to investigate the possibility of targeting thrombomodulin in patients with DN. Glomerular thrombomodulin was analyzed together with the number of glomerular macrophages in 90 autopsied diabetic cases with DN, 55 autopsied diabetic cases without DN, and 37 autopsied cases without diabetes or kidney disease. Thrombomodulin mRNA was measured in glomeruli microdissected from renal biopsies from patients with DN and nondiabetic controls. Finally, glomerular thrombomodulin was measured in diabetic mice following treatment with the selective endothelin A receptor (ETAR) blocker, atrasentan. In diabetic patients, glomerular thrombomodulin expression was increased at the mRNA level, but decreased at the protein level, compared with nondiabetic controls. Reduced glomerular thrombomodulin was associated with an increased glomerular influx of macrophages. Blocking the ETAR with atrasentan restored glomerular thrombomodulin protein levels in diabetic mice to normal levels. The reduction in glomerular thrombomodulin in diabetes likely serves as an early proinflammatory step in the pathogenesis of DN. Thrombomodulin protein may be cleaved under diabetic conditions, leading to a compensatory increase in transcription. The nephroprotective effects of ETAR antagonists in diabetic patients may be attributed to the restoration of glomerular thrombomodulin.

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 Glycocalyx Hyaluronan: Regulation and Role in Prevention of Diabetic Complications.

The endothelial glycocalyx is critically involved in vascular integrity and homeostasis, by regulating vascular permeability, regulating mechanotransduction, and reducing inflammation and coagulation. The turnover of the glycocalyx is dynamic to fine-tune these processes. This is in particular true for its main structural component, hyaluronan (HA). Degradation and shedding of the glycocalyx by enzymes, such as hyaluronidase 1 and hyaluronidase 2, are responsible for regulation of the glycocalyx thickness and hence access of circulating cells and factors to the endothelial cell membrane and its receptors. This degradation process will at the same time also allow for resynthesis and adaptive chemical modification of the glycocalyx. The (re)synthesis of HA is dependent on the availability of its sugar substrates, thus linking glycocalyx biology directly to cellular glucose metabolism. It is therefore of particular interest to consider the consequences of dysregulated cellular glucose in diabetes for glycocalyx biology and its implications for endothelial function. This review summarizes the metabolic regulation of endothelial glycocalyx HA and its potential as a therapeutic target in diabetic vascular complications.