Endothelial Fenestrations Research Articles

Fenestrated Endothelial Cells across Organs: Insights into Kidney Function and Disease.

In the human body, the vascular system plays an indispensable role in maintaining homeostasis by supplying oxygen and nutrients to cells and organs and facilitating the removal of metabolic waste and toxins. Blood vessels-the key constituents of the vascular system-are composed of a layer of endothelial cells on their luminal surface. In most organs, tightly packed endothelial cells serve as a barrier separating blood and lymph from surrounding tissues. Intriguingly, endothelial cells in some tissues and organs (e.g., choroid plexus, liver sinusoids, small intestines, and kidney glomerulus) form transcellular pores called fenestrations that facilitate molecular and ionic transport across the vasculature and mediate immune responses through leukocyte transmigration. However, the development and unique functions of endothelial cell fenestrations across organs are yet to be fully uncovered. This review article provides an overview of fenestrated endothelial cells in multiple organs. We describe their development and organ-specific roles, with expanded discussions on their contributions to glomerular health and disease. We extend these discussions to highlight the dynamic changes in endothelial cell fenestrations in diabetic nephropathy, focal segmental glomerulosclerosis, Alport syndrome, and preeclampsia, and how these unique cellular features could be targeted for therapeutic development. Finally, we discuss emerging technologies for in vitro modeling of biological systems, and their relevance for advancing the current understanding of endothelial cell fenestrations in health and disease.

Deletion of the endothelial glycocalyx component endomucin leads to impaired glomerular structure and function.

Endomucin (EMCN), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of vascular endothelial growth factor (VEGF) activity through modulating VEGF receptor 2 (VEGFR2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. Global EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was employed to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, while urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, albumin, and α-smooth muscle actin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a non-invasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA-seq. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis of up- and down-regulated genes. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. EMCN -/- mice were viable with no gross anatomical defects in kidneys. The EMCN -/- mice exhibited increased infiltration of CD45 + cells, with an increased proportion of Ly6G high Ly6C high myeloid cells and higher VCAM-1 expression. EMCN -/- mice displayed albuminuria with increased albumin in the Bowman's space compared to the EMCN +/+ littermates. Glomeruli in EMCN -/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA-seq of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK/ERK pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. Our study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.

A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

#246 Understanding the impact of antiangiogenic drug-induced proteinuria on kidney function in cancer patients

Abstract Background and Aims Proteinuria is a common adverse event associated with the use of vascular endothelial growth factor (VEGF) inhibitor drugs, which are widely employed in oncology for treating various tumors. The incidence of proteinuria of all degrees ranges from 10% to 90%, with proteinuria in the nephrotic range occurring in 1-5% of cases. VEGF plays a crucial role in maintaining the integrity of the glomerular capillary wall. Podocytes, constitutively express VEGF, and VEGF receptors are present on both the surface of glomerular capillary endothelial cells and podocytes. Any disruption in the podocyte-endothelial VEGF axis can lead to the loss of endothelial fenestrations in glomerular capillaries, proliferation of glomerular endothelial cells, loss of podocytes, and subsequent proteinuria. The most common pathological findings include focal segmental glomerulosclerosis and minimal change nephropathy, typically associated with tyrosine kinase inhibitors (TKIs), as well as thrombotic microangiopathy (TMA), more frequently observed in pts treated with anti VEGF. Proteinuria is a recognized risk factor for progressive renal damage and end-stage renal disease. However, it's worth noting that the average treatment duration for patients on anti-angiogenic therapy is considerably shorter than the time indicated by clinical studies as necessary to induce renal damage from proteinuria in healthy individuals. However, the long-term impact of drug-induced proteinuria on renal function remains uncertain. The aim of the study is to investigate the association between proteinuria induced by anti-VEGF/VEGFR drugs and its long-term effects on renal function in cancer patients. Method A retrospective study was conducted on 124 patients undergoing anti-VEGF/VEGFR therapy for ≥6 months. 45% of the patients enrolled in the study were on TKI monotherapy, 36% received TKI plus immune checkpoint inhibitors (ICIs), 10% received anti- VEGF treatment, 7% received TKI + chemotherapy (CT), and 2% received anti-VEGF + ICIs. Among patients, 51% were undergoing treatment for kidney cancer, 25% for thyroid cancer, 14% for colon cancer, 7% for liver cancer, and 5% for lung cancer. The study assessed the time course of estimated glomerular filtration rate (eGFR), the influence of baseline eGFR on subsequent variations, the association between proteinuria and renal function, and the identification of risk factors for proteinuria or renal failure. Proteinuria was quantified as proteinuria/24 h. Results Among the 124 patients, 53 developed proteinuria <1 g/24 h, 41 between 1-3 g/24 h, and 30 >3 g/24 h. Specific data are outlined in Table 1. A trend toward a reduction in eGFR of −5 ml/min/1.73 m2 was observed in all patients after 2 years of treatment. The high proteinuria group exhibited a greater percentage change in eGFR compared to the other groups (−1.5% vs −8.5% vs −12.3%) as depicted in Fig. 1A, along with a time-dependent decrease in eGFR. There was no significant difference in the development of eGFR <30 ml/min/1.73 m2 between groups, and no patients permanently discontinued treatment due to nephrotoxicity. No differences in eGFR reduction were noted among the three groups categorized by baseline eGFR (Fig. 1B). Importantly, renal function after the end of therapy was found to be reversible. Conclusion The study did not find a correlation between the degree of proteinuria secondary to anti-VEGF/VEGFR and significant reductions in renal function. Therefore, treatment can be continued irrespective of the degree of proteinuria. Clinical decisions regarding the continuation of therapy with these drugs should weigh the benefits in terms of survival against the potential risks of toxicity, including a modest reduction in renal glomerular filtration rate.

Correlation of light and electron microscopic morphometric parameters of glomerular capillaries with serum creatinine and proteinuria

ABSTRACT Waste products in the bloodstream are filtered by the glomerular capillaries in the kidneys and excreted into the urine. When making a differential diagnosis of kidney diseases, structural assessment of glomeruli using histological, ultrastructural, and immunological studies is crucial. This study assessed the microscopic and ultrastructural morphometric parameters of glomerular capillaries and examined their correlation with serum creatinine and proteinuria. A total of 60 kidney biopsy cases received by the transmission electron microscope (TEM) laboratory for diagnosis were included in the study. Toluidine blue stained 300 nm thick sections of TEM tissue blocks were scanned for glomerular morphometry by a whole slide imaging system, and the estimation of Bowman’s capsule (BC) area, glomerular capillary lumen diameter (GCLD), glomerular capillary density (GCD), glomerular capillary surface area density (GCSA), and percentage of glomerular capillary lumen space (%GCLS) was performed with QuPath software. TEM images of 70 nm thick sections were used for the evaluation of endothelial fenestration diameter (EFD), glomerular basement membrane (GBM) thickness, and podocyte foot process (PFP) effacement. Proteinuria and serum creatinine showed positive correlations with GBM thickness and PFP effacement. Negative correlations of serum creatinine were observed with EFD, %GCLS, and GCSA. Hence, glomerular filtration is greatly affected by the total area of the glomerular capillary surface and structural changes of GBM. Reduction of glomerulus filtration due to foot process effacement and thickening of GBM results in damage to the filtration barrier leading to the leakage of plasma protein into urine.

Induction of Fenestrae in Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Disease Modeling.

The endothelial linings of capillaries, such as those in the kidney and small intestines, possess fenestrae that facilitate fluid and exchange of small molecules. Alterations in the size and number of endothelial fenestrae have been implicated in the pathogenesis of various diseases. The re-creation of fenestrated endothelium using human induced pluripotent stem cells (hiPSCs) provides a promising avenue to investigate the involvement of fenestrae in disease mechanisms and pharmacodynamics. In this project, we aim to induce the formation of fenestrae in nonfenestrated hiPSCs-derived endothelial cells (hiPSC-ECs). Vascular endothelial growth factor A (VEGFA) and phorbol myristate acetate (PMA) were used as inducers of fenestrae in hiPSC-ECs. The assessment of fenestrae formation included gene-expression analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and immunofluorescent staining. Endothelial monolayer functionality was evaluated by dextran permeability assays. Stimulation with VEGFA and PMA significantly induced expression of the diaphragmed fenestrae-associated marker, plasmalemmal vesicle-associated protein (PLVAP), in hiPSC-ECs at the mRNA, and protein levels. SEM analysis revealed VEGFA- and PMA-induced fenestrae structures on the cell membrane of hiPSC-ECs. The increased membrane localization of PLVAP visualized by TEM and immunofluorescent staining supported these findings. The induced fenestrated endothelium in hiPSC-ECs demonstrated selective passage of small solutes across a confluent monolayer with intact cell junctions, confirming functional competence. In conclusion, we present a novel methodology for inducing and regulating fenestrated endothelium in hiPSC-ECs. This innovative approach paves the way for the development of fenestrated microvasculature in human organ-on-a-chip systems, enabling complex disease modeling and physiologically relevant investigations of pharmacodynamics.

Glycolytic enzyme PFKFB3 regulates sphingosine 1-phosphate receptor 1 in proangiogenic glomerular endothelial cells under diabetic condition.

Glomerular angiogenesis is a characteristic feature of diabetic nephropathy (DN). Enhanced glycolysis plays a crucial role in angiogenesis. The present study was designed to investigate the role of glycolysis in glomerular endothelial cells (GECs) in a mouse model of DN. Mouse renal cortex and isolated glomerular cells were collected for single-cell and RNA sequencing. Cultured GECs were exposed to high glucose in the presence (proangiogenic) and absence of a vascular sprouting regimen. MicroRNA-590-3p was delivered by lipofectamine in vivo and in vitro. In the present study, a subgroup of GECs with proangiogenic features was identified in diabetic kidneys by using sequencing analyses. In cultured proangiogenic GECs, high glucose increased glycolysis and phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) protein expression, which were inhibited by overexpressing miRNA-590-3p. Mimics of miRNA-590-3p also increased receptor for sphingosine 1-phosphate (S1pR1) expression, an angiogenesis regulator, in proangiogenic GECs challenged with high glucose. Inhibition of PFKFB3 by pharmacological and genetic approaches upregulated S1pR1 protein in vitro. Mimics of miRNA-590-3p significantly reduced migration and angiogenic potential in proangiogenic GECs challenged with high glucose. Ten-week-old type 2 diabetic mice had elevated urinary albumin levels, reduced renal cortex miRNA-590-3p expression, and disarrangement of glomerular endothelial cell fenestration. Overexpressing miRNA-590-3p via perirenal adipose tissue injection restored endothelial cell fenestration and reduced urinary albumin levels in diabetic mice. Therefore, the present study identifies a subgroup of GECs with proangiogenic features in mice with DN. Local administration of miRNA-590-3p mimics reduces glycolytic rate and upregulates S1pR1 protein expression in proangiogenic GECs. The protective effects of miRNA-590-3p provide therapeutic potential in DN treatment.NEW & NOTEWORTHY Proangiogenetic glomerular endothelial cells (GECs) are activated in diabetic nephropathy. High glucose upregulates glycolytic enzyme phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) in proangiogenetic cells. PFKFB3 protects the glomerular filtration barrier by targeting endothelial S1pR1. MiRNA-590-3p restores endothelial cell function and mitigates diabetic nephropathy.

The unique structural and functional characteristics of glomerular endothelial cell fenestrations and their potential as a therapeutic target in kidney disease.

Glomerular endothelial cell (GEnC) fenestrations are a critical component of the glomerular filtration barrier. Their unique nondiaphragmed structure is key to their function in glomerular hydraulic permeability, and their aberration in disease can contribute to loss of glomerular filtration function. This review provides a comprehensive update of current understanding of the regulation and biogenesis of fenestrae. We consider diseases in which GEnC fenestration loss is recognized or may play a role and discuss methods with potential to facilitate the study of these critical structures. Literature is drawn from GEnCs as well as other fenestrated cell types such as liver sinusoidal endothelial cells that most closely parallel GEnCs.

Prognostic role of glomerular electron microscopy lesions in IgA nephropathy: "the devil is in the details".

Transmission electron microscopy enables examination of ultrastructural glomerular changes; while this tool has already been appliedin IgA nephropathy (IgAN), limited information exists on the prognostic valuein this disease. We aimed to systematically investigate ultrastructural lesions and assess their role in predicting the evolution of IgA nephropathy toend-stage kidney disease. A single-center retrospective study was performed on 107 consecutive IgAN patients (median age 42years, 67% male, estimated glomerular filtration rate 46mL/min, proteinuria 1.0g/g) between 2010 and 2015, who were followed-up until end-stage kidney disease, death, or end of study (January 2021). A pathologist evaluated the Mesangial hypercellularity (M), Endocapillary hypercellularity (E), Segmental glomerulosclerosis (S), and Tubular atrophy/interstitial fibrosis-Crescents (C) (MEST-C) score and transmission electron microscopy lesions according to a comprehensive protocol that encompassed all glomerular structures. Patients were followed up for a median of 7.1years; 32 (43%) reached end-stage kidney disease. Patients who reached kidney failure had higher comorbidity score, more frequent arterial hypertension, lower estimated glomerular filtration rate, and higher MEST-C score. In terms of transmission electron microscopy lesions, patients who progressed to end-stage kidney disease had more frequent podocyte activation, effacement, and presence of microvilli; more frequent signs of endothelial cell activation and fenestration; higher mesangial cell proliferation. In theunivariate Cox proportional hazard regression, higher MEST-C score and lesions detected bytransmission electron microscopy in podocytes, endothelial cells, and mesangial cell proliferation were associated with shorter kidney survival time. In themultivariate Cox proportional hazard regression, only higher MEST-C score, presence of podocytes with microvilli, and mesangial cell proliferation were associated with end-stage kidney disease. This study shows that, besides the MEST-C score, the presence of podocytes with microvilli and mesangial cell proliferation are associated with poor kidney survival in IgAN patients, highlighting the prognostic value of lesions detected bytransmission electron microscopy.

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis

The median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism.

Delivery of Drugs into Cancer Cells Using Antibody-Drug Conjugates Based on Receptor-Mediated Endocytosis and the Enhanced Permeability and Retention Effect.

Innumerable people worldwide die of cancer every year, although pharmaceutical therapy has actualized many benefits in human health. For background, anti-cancer drug development is difficult due to the multifactorial pathogenesis and complicated pathology of cancers. Cancer cells excrete hydrophobic low-molecular anti-cancer drugs by overexpressed efflux transporters such as multiple drug resistance 1 (MDR1) at the apical membrane. Mutation-driven drug resistance is also developed in cancer. Moreover, the poor distribution of drug to cancer cells is a serious problem, because patients suffer from off-target side effects. Thus, highly selective and effective drug delivery into solid cancer cells across the membrane should be established. It is known that substances (10-100 nm in diameter) such as monoclonal antibodies (mAbs) (approximately 14.2 nm in diameter) or nanoparticles spontaneously gather in solid tumor stroma or parenchyma through the capillary endothelial fenestration, ranging from 200-2000 nm, in neovasculatures due to the enhanced permeability and retention (EPR) effect. Furthermore, cancer antigens, such as HER2, Nectin-4, or TROP2, highly selectively expressed on the surface of cancer cells act as a receptor for receptor-mediated endocytosis (RME) using mAbs against such antigens. Thus, antibody-drug conjugates (ADCs) are promising anti-cancer pharmaceutical agents that fulfill accurate distribution due to the EPR effect and due to antibody-antigen binding and membrane permeability owing to RME. In this review, I introduce the implementation and possibility of highly selective anti-cancer drug delivery into solid cancer cells based on the EPR effect and RME using anti-cancer antigens ADCs with payloads through suitable linkers.

Notch Pathway Driven Delayed Dedifferentiation of Liver Sinusoidal Endothelium Impacts Liver-Directed Gene Transfer in Hemophilia-a Mice

Background: Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which is costly, inconvenient, and does not fully prevent bleeding. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategies for treating hemophilia have emerged. The Factor VIII null mouse (F8TKO mice) has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver sinusoidal endothelial cells (LSECs). Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. Methods: In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in F8TKO mouse using recombinant adeno associated virus (AAV)8 vector. AAV8-TBG-GFP was administered through IP and IV, and GFP expression was analyzed in the liver 15- and 30-days post injection. Results: Immunofluorescence of GFP at day 15 and day 30 post injection revealed significantly fewer GFP+ cells (p=0.04) in the liver of F8TKO compared with control mice post AAV8-GFP injection. This was further validated by ELISA assays and western blot for GFP. Moreover, Caspase-3 staining revealed significant enrichment (p=0.05) in the liver of F8TKO mice compared with control mice after AAV8-GFP administration at both the 15- and 30-day time points suggestive of increased LSEC apoptosis post AAV8-GFP injection. To further confirm apoptosis of LSECs, we next used liver intravital imaging to visualize LSECs in F8TKO mice at baseline and upon AAV8-GFP administration. TXR-dextran and AF488-anti-CD31 antibody were IV administered to visualize hepatic blood flow and LSECs, respectively. At baseline, both control and F8TKO mice showed normal blood flow and enriched expression of AF488-anti-CD31. However, there were fewer AF488-anti-CD31+endothelial cells in F8TKO than control mice at day 15 post-AAV8-GFP treatment (p=0.01), suggestive of apoptosis of LSECs in F8TKO mice post-AAV8-GFP administration. Scanning electron microscopy revealed dramatic reduction (p=0.01) in endothelial fenestration in the liver of F8TKO as compared with control mouse liver. The absence of fenestrae has been associated with LSEC capillarization, which promotes basement membrane formation while decreasing permeability. Remarkably, we found significant upregulation of LSEC markers associated with capillarization, including CD31, vascular endothelial growth factor (VEGF), ICAM1 (Intercellular Adhesion Molecule-1), stabilin2, ID1 (Inhibitor of DNA Binding-1), Gata4, and Ehd3 (EH Domain Containing-3) in F8TKO mice by qRT-PCR) and western blot. Mechanistically we show significant upregulation of Notch pathway components (Notch 1, 3, and Jagged 2) by qRT-PCR and western in the liver of F8TKO mice. IHC analysis showed significant upregulation of notch 3 early in development in F8TKO mice leading to pseudocapillarization of LSECs. Moreover, we also show that notch activation regulates nitric oxide/soluble guanylyl cyclase signaling by upregulating downstream targets (NOS-1, 2, 3, PDE1a, b, c and 10B) in LSECs which directly controls the development of LSEC fenestration. Conclusion: In conclusion, we demonstrate molecular characterization of LSEC-mediated liver-directed gene transfer in a null hemophilia A mouse model, which is critical for understanding the efficacy and stability of AAV-FVIII gene-based hemophilia treatment. Work is currently underway to understand how absence of FVIII can affect the LSECs mediated hepatic gene transfer in Hemophilia-A mice and how modulation of Notch and nitric oxide/sGC signaling can manipulate LSEC fenestration and improve the efficiency of liver directed gene transfer.

Carthami flos extract against carbon tetrachloride-induced liver fibrosis via alleviating angiogenesis in mice

BackgroundAngiogenesis is a pathological phenomenon contribute to the development of chronic liver diseases, and anti-angiogenic therapy is an effective strategy to alleviate liver fibrosis. Carthami flos, a medicinal and edible herb, has the effects of improving blood circulation and regulating angiogenesis. However, the anti-angiogenic effect of Carthami flos in liver fibrosis remains unknown. MethodsWe investigated the protective effect and therapeutic mechanism of Carthami flos extract (CFE) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. The liver injury and collagen deposition were observed and evaluated by conducting HE, Masson, and Sirius red staining, testing the serum biochemical indexes (ALT, AST, ALP, γ-GT), and measuring the contents of HYP and four indexes of liver fiber (Col-IV, LN, HA, PC-III). Simultaneously, the expressions of α-SMA and Collagen-I were detected to determine the activation of hepatic stellate cells (HSCs). Subsequently, we measured the expressions of angiogenesis-related proteins such as PDGFRB, ERK1/2, p-ERK1/2, MEK, p-MEK, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, and the mRNA levels of PDGFRB and VEGFA. Additionally, immunofluorescence staining and RT-qPCR assays were carried out to ascertain the expressions of continuous endothelial markers CD31, CD34 and vWF, and scanning electron microscope analysis was performed to observe the number of sinusoidal endothelial fenestrations. ResultsHerein, we found that CFE could significantly reduce liver injury and collagen deposition, like the same effect of colchicine. CFE significantly alleviated CCl4-induced liver injury and fibrosis, mainly manifested by reducing the levels of ALT, AST, ALP and γ-GT and decreasing the contents of HYP, Col-IV, LN, HA and PC-III. Additionally, CCl4 promoted the activation of HSCs by increasing the expressions of α-SMA and Collagen-I, while CFE could rectify the condition. Moreover, CFE treatment prevented the CCl4-induced the up-regulation of PDGFRB, p-MEK, p-ERK1/2, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, suggesting that CFE might provide the protection against abnormal angiogenesis. In the meantime, the gradual disappearance of sinusoidal capillarization after CFE treatment was supported by the decreased the contents of CD31, CD34 and vWF, as well as the increased number of sinusoidal endothelial fenestrae. ConclusionIn this study, the reduction of collagen deposition, the obstruction of HSCs activation, the inactivation of angiogenic signaling pathways and the weakening of hepatic sinusoidal capillarization jointly confirmed that CFE might be promising to resist angiogenesis in liver fibrosis via the PDGFRB/ERK/HIF-1α and VEGFA/AKT/eNOS signaling pathways. Nevertheless, as a potential therapeutic drug, the deeper mechanism of Carthami flos still needs to be further elucidated.

ADAMTS18+ villus tip telocytes maintain a polarized VEGFA signaling domain and fenestrations in nutrient-absorbing intestinal blood vessels

The small intestinal villus tip is the first point of contact for lumen-derived substances including nutrients and microbial products. Electron microscopy studies from the early 1970s uncovered unusual spatial organization of small intestinal villus tip blood vessels: their exterior, epithelial-facing side is fenestrated, while the side facing the villus stroma is non-fenestrated, covered by pericytes and harbors endothelial nuclei. Such organization optimizes the absorption process, however the molecular mechanisms maintaining this highly specialized structure remain unclear. Here we report that perivascular LGR5+ villus tip telocytes (VTTs) are necessary for maintenance of villus tip endothelial cell polarization and fenestration by sequestering VEGFA signaling. Mechanistically, unique VTT expression of the protease ADAMTS18 is necessary for VEGFA signaling sequestration through limiting fibronectin accumulation. Therefore, we propose a model in which LGR5+ ADAMTS18+ telocytes are necessary to maintain a “just-right” level and location of VEGFA signaling in intestinal villus blood vasculature to ensure on one hand the presence of sufficient endothelial fenestrae, while avoiding excessive leakiness of the vessels and destabilization of villus tip epithelial structures.

Reduced Glomerular Filtration in Diabetes Is Attributable to Loss of Density and Increased Resistance of Glomerular Endothelial Cell Fenestrations.

Glomerular endothelial cell (GEnC) fenestrations are recognized as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease. We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular Kf and GFR in diabetic mice (BTBR ob-/ob- ). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explored its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells. Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knockdown b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation. This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease.

MO260: Prognostic Role of Electron Microscopy Lesions in IGA Nephropathy

Abstract BACKGROUND AND AIMS The progression of immunoglobulin A nephropathy (IgAN) is currently assessed using the Oxford MEST-C score, which uses five indicators (mesangial and endocapillary hypercellularity, segmental sclerosis, interstitial fibrosis/tubular atrophy and the presence of crescents). However, little is known about the prognostic role of the glomerular lesions assessed by transmission electron microscopy (EM). METHOD We performed a unicentric retrospective study on 107 consecutive IgAN patients {age 42 [interquartile range (IQR) 33–54] years}, 67% male, eGFR 46 (IQR 33.3–65.2) mL/min, proteinuria 1.0 (IQR 0.4–2.1) g/g creatinine)—kidney biopsy-proven—between 2010 and 2015. Patients were followed until end-stage kidney disease (ESKD, dialysis or renal transplantation), death or end of study (January 2021), whichever came first. For each biopsy specimen, light microscopy, immunofluorescence and EM were routinely performed. One pathologist (GTB) reviewed independently the slides without knowledge of the original biopsy interpretation and assessed for MEST-C score and EM lesions according to the Mayo Clinic/Renal Pathology Society Consensus in podocytes, endothelial cells, mesangium and glomerular basement membrane (Table 1). RESULTS Patients were followed for a median of 7.1 (95% CI 6.7–7.5) years. The patients who reached ESKD—32 (43%)—had a higher Charlson comorbidity score [CCS 2 (IQR 2–4) versus 1 (IQR 0–3), P = .01)], arterial hypertension more often [88% versus 64%, P = .01)], lower eGFR [33.7 (IQR 22.4–44.3) versus 56.3 (IQR 39.4–72.5) mL/min, P < .001] and higher MEST-C score [2 (IQR 2–4) versus 1 (IQR 1–2), P < .001]. There were no differences regarding proteinuria, hematuria and treatment. In terms of EM lesions, patients who experienced ESKD had more frequent podocyte activation (47% versus 27%, P < 0.01), effacement (100% versus 83%, P = .01) and presence of microvilli (69% versus 35%, P = .001); more often endothelial cells activation (47% versus 27%, P = .04) and fenestration (56% versus 29%, P < 0.01); higher mesangial cells proliferation (91% versus 64%, P < .01). Mean kidney survival time for the entire cohort was 8.2 (95% CI 7.4–8.9) years. In univariate Cox proportional hazard (CPH) regression higher MEST-C score and EM lesions in podocytes, endothelial cells and mesangial cell proliferation were associated with a shorter kidney survival time (Table 1). However, in the multivariate CPH adjusted for CCS, arterial hypertension, eGFR and treatment, only higher MEST-C score, presence of podocytes with microvilli and mesangial cell proliferation were associated with ESKD (Table 1). CONCLUSION To the best of our knowledge, this is the first study to evaluate in depth the prognostic value of EM lesions in IgAN patients in a reasonable large cohort with an appropriate follow-up time. Besides the MEST-C score, we found that the presence of podocytes with microvilli and mesangial cell proliferation are associated with poor kidney survival.

EMCN knockout leads to increased monocyte infiltration in kidney and albuminuria in mice

Endomucin (EMCN), a type I integral membrane glycoprotein, is specifically expressed in endothelium and enriched in kidney glomerular endothelium. We have previously reported that under resting conditions EMCN functions as an anti‐inflammatory molecule, preventing leukocyte‐endothelial cell adhesion and that EMCN plays an important role in modulating VEGFR2 signaling. The goal of this study is to characterize the phenotype of the kidney in the first‐reported EMCN knockout mouse model. EMCN−/−mice (with a EGFP tag for the knockout allele) were obtained by crossing EMCN‐floxed mice, generated in conjunction with Cyagen Biosciences, with the ROSA26‐Cre mice. The mRNA level of EMCN, CD31, CD34, E‐selection, VCAM‐1, and ICAM‐1 were quantified by reverse transcription PCR and semi‐quantitative PCR using total RNA isolated from the adult mouse kidneys. The mRNA level of EMCN in EMCN−/−mice were not detectable (n>4, p<0.0001) compared to EMCN+/+. Renal CD34 at mRNA level was decreased (fold change, 0.68±0.09 vs 1, n=4, p<0.05) and VCAM‐1 was increased (fold change, 2.2 ±0.45 vs 1, n=4, p<0.01) in EMCN−/− kidneys compared to wildtype (wt) controls. CD31, E‐selection, and ICAM‐1 mRNA levels were unchanged between EMCN−/−and wildtype kidneys. Kidneys from adult EMCN+/+ and EMCN−/− mice were collected, fixed, and sectioned. Expression and localization of EMCN, eGFP, CD45 and podocin in adult kidneys were examined by immunohistochemistry and quantified, which revealed a significant increase in CD45+ cell infiltration in EMCN−/− mice compared to EMCN+/+ mice (72.50±8.13 cells/mm2 vs 39.67±4.91 cells/ mm2, n=4, p<0.001). To characterize the renal inflammatory cell composition, freshly collected kidneys from EMCN+/+ and EMCN−/− were enzymatically digested and the leukocyte markers CD45, CD3, CD4, CD19, CD11b, Ly6G and Ly6C were examined by flow cytometry to identify the proportion of myeloid populations. Flow cytometry analysis suggested a trend of increased CD45+ cells in the kidney from EMCN−/−mice compared to wildtype (n=9, p=0.07), and revealed a significant increase in the percentage of Ly6GhighLy6Chighmyeloid cells in EMCN−/−compared to wildtype kidneys (1.6±0.23 vs 1.01±0.18, n=9, p<0.05). Sporadic albuminuria was detected through Coomassie staining in spot urine samples of EMCN−/− mice. Ultrastructural morphology of glomeruli, examined from both EMCN+/+ and EMCN−/− through transmission electron microscopy, showed the effacement of podocyte foot processes and disorganized endothelial cell fenestrations in glomerular capillaries of EMCN−/− mice compared to wt. The result indicates EMCN plays a key role in the prevention of inflammatory cells infiltration in kidney, presumably by blocking leukocyte adhesion, and is also involves in maintaining normal glomerular filtration barrier structure and function, potentially via its role in VEGFR2 signaling.

Role of glomerular endothelial cell fenestrations

Role of glomerular endothelial cell fenestrations

Thrombopoietin accumulation in hepatocytes induces a decrease in its serum levels in a sinusoidal obstruction syndrome model.

Sinusoidal obstruction syndrome (SOS) is a type of fatal hepatic injury, which predominantly occurs following exposure to drugs, such as oxaliplatin, or bone marrow transplantation. Extravasated platelet aggregation (EPA) plays an important role in the development of SOS in rat and mouse models. Furthermore, platelets invading the space of Disse adhere to hepatocytes and are phagocytized in patients with SOS. Aging platelets and platelets in patients with sepsis are phagocytized by hepatocytes through Ashwell‑Morell receptors, and thrombopoietin (TPO) is produced by the JAK2‑STAT3 signaling pathway. The purpose of the present study was to examine the significance of TPO as a biomarker of SOS. SOS was induced in Crl:CD1(ICR) female mice by intraperitoneal administration of monocrotaline (MCT). TPO levels were measured in the serum and liver tissue. Pathological and immunohistochemical studies of the liver were performed to analyze the expression levels of TPO. TPO mRNA expression levels were measured using reverse transcription‑quantitative PCR. In the SOS model, the platelet counts in peripheral blood samples were significantly decreased at 24and48h after MCT treatment as compared with that at 0h. In addition, a pathological change in hepatic zone3 was observed in the SOS model group. Furthermore, the protein levels of TPO in liver tissue were significantly increased in the SOS model group compared with those in the control group, which was confirmed by immunohistochemistry. By contrast, serum TPO protein levels were significantly decreased in the SOS model group compared with those in the control group. These results indicated that EPA may induce sinusoidal endothelial fenestration in a mouse model of SOS, preventing TPO from translocating into the blood. In conclusion, serum TPO levels may be reduced in a mouse model of SOS owing to the accumulation in hepatocytes, suggesting that TPO could be a useful biomarker of SOS.

From fixed-dried to wet-fixed to live – comparative super-resolution microscopy of liver sinusoidal endothelial cell fenestrations

Abstract Fenestrations in liver sinusoidal endothelial cells (LSEC) are transcellular nanopores of 50–350 nm diameter that facilitate bidirectional transport of solutes and macromolecules between the bloodstream and the parenchyma of the liver. Liver diseases, ageing, and various substances such as nicotine or ethanol can negatively influence LSECs fenestrations and lead to defenestration. Over the years, the diameter of fenestrations remained the main challenge for imaging of LSEC in vitro. Several microscopy, or rather nanoscopy, approaches have been used to quantify fenestrations in LSEC to assess the effect of drugs and, and toxins in different biological models. All techniques have their limitations, and measurements of the “true” size of fenestrations are hampered because of this. In this study, we approach the comparison of different types of microscopy in a correlative manner. We combine scanning electron microscopy (SEM) with optical nanoscopy methods such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy. In addition, we combined atomic force microscopy (AFM) with SEM and STED, all to better understand the previously reported differences between the reports of fenestration dimensions. We conclude that sample dehydration alters fenestration diameters. Finally, we propose the combination of AFM with conventional microscopy that allows for easy super-resolution observation of the cell dynamics with additional chemical information that can be traced back for the whole experiment. Overall, by pairing the various types of imaging techniques that provide topological 2D/3D/label-free/chemical information we get a deeper insight into both limitations and strengths of each type microscopy when applied to fenestration analysis.