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Rationale & Objective: Individuals with chronic kidney disease (CKD) are at increased risk of morbidity and mortality, particularly as they progress to kidney failure. Identifying circulating proteins that underlie kidney failure development may guide the discovery of new targets for intervention. Study Design: Prospective cohort. Setting & Participants: 703 African American Study of Kidney Disease and Hypertension (AASK) and 434 Boston Kidney Biopsy Cohort (BKBC) participants with baseline proteomics data. Exposures: Circulating proteins measured using SomaScan. Outcomes: Kidney failure, defined as dialysis initiation or kidney transplantation. Analytical Approach: Using adjusted Cox models, we studied associations of 6,284 circulating proteins with kidney failure risk separately in AASK and BKBC and meta-analyzed results. We then performed gene set enrichment analyses to identify underlying perturbations in biological pathways. In separate data sets with kidney-tissue level gene expression, we ascertained dominant regions of expression and correlated kidney tubular gene expression with fibrosis and estimated glomerular filtration rate (eGFR). Results: Over median follow-up periods of 8.8 and 3.1 years, 210 AASK (mean age: 55 years, 39% female, mean GFR: 46 mL/min/1.73 m2) and 115 BKBC (mean age: 54 years, 47% female, mean eGFR: 51 mL/min/1.73 m2) participants developed kidney failure, respectively. We identified 143 proteins that were associated with incident kidney failure, of which only 1 (Testican-2) had a lower risk. Notable proteins included those related to vascular permeability (endothelial cell-selective adhesion molecule), glomerulosclerosis (ephrin-A1), glomerular development (ephrin-B2), intracellular sorting/transport (vesicular integral-membrane protein VIP36), podocyte effacement (pigment epithelium-derived factor), complement activation (complement decay-accelerating factor), and fibrosis (ephrin-A1, ephrin-B2, and pigment epithelium-derived factor). Gene set enrichment analyses detected overrepresented pathways that could be related to CKD progression, such as ephrin signaling, cell-cell junctions, intracellular transport, immune response, cell proliferation, and apoptosis. At the kidney level, glomerular expression predominated for genes corresponding to circulating proteins of interest, and several gene expression levels were correlated with eGFR and/or fibrosis. Limitations: Possible residual confounding. Conclusions: Multimodal data identified proteins and pathways associated with the development of kidney failure. Plain-Language Summary: Circulating proteins that underlie the development of kidney failure may be new targets for treatment. In the current study of adults with chronic kidney disease, we evaluated over 6,000 proteins detected in blood and found more than 100 proteins whose levels were associated with new-onset kidney failure. Further investigation using gene expression data showed that the genes encoding these proteins were expressed in the kidney and involved in pathways of immune responses as well as cell signaling, structure, transport, and survival.

A female infant born with a gestational age of 35 weeks and birth weight of 2500 g was referred for ophthalmic examination on the second postnatal day. Bilateral venous dilatation and arterial tortuosity, severe extraretinal fibrovascular proliferation, and peripheral ischemia were detected. Fluorescein angiography showed profoundly delayed arteriovenous transit and peripheral avascularity. Both eyes were treated with diode laser photocoagulation and bevacizumab injection. Cranial magnetic resonance imaging (MRI) revealed hydrocephalus, ventricular dilatation, and cerebral atrophy. Her family history revealed that the patient’s brother presented to the ophthalmology outpatient clinic at postnatal 3 months with inoperable total retinal detachment and similar cranial MRI findings. No systemic or ocular findings were detected in the parents. A recent study showed that in 13 cases, including our patients, bi-allelic variants in the ESAM gene lead to a new neurodevelopmental disease whose main clinical features include impaired speech and language development, seizures, varying degrees of spasticity, ventriculomegaly, intracranial hemorrhage, and developmental delay/mental disability. Newborn siblings of children with serious pathological retinal findings should undergo a detailed ophthalmic examination as soon as possible after birth to prevent total retinal detachment, even without a diagnosis of specific inherited retinal vascular diseases. Further investigations performed in collaboration with an international network may reveal more candidate gene variants possibly related to retinopathy of prematurity-like ophthalmological findings such as extraretinal fibrovascular proliferation.

A female infant born with a gestational age of 35 weeks and birth weight of 2500 g was referred for ophthalmic examination on the second postnatal day. Bilateral venous dilatation and arterial tortuosity, severe extraretinal fibrovascular proliferation, and peripheral ischemia were detected. Fluorescein angiography showed profoundly delayed arteriovenous transit and peripheral avascularity. Both eyes were treated with diode laser photocoagulation and bevacizumab injection. Cranial magnetic resonance imaging (MRI) revealed hydrocephalus, ventricular dilatation, and cerebral atrophy. Her family history revealed that the patient's brother presented to the ophthalmology outpatient clinic at postnatal 3 months with inoperable total retinal detachment and similar cranial MRI findings. No systemic or ocular findings were detected in the parents. A recent study showed that in 13 cases, including our patients, bi-allelic variants in the ESAM gene lead to a new neurodevelopmental disease whose main clinical features include impaired speech and language development, seizures, varying degrees of spasticity, ventriculomegaly, intracranial hemorrhage, and developmental delay/mental disability. Newborn siblings of children with serious pathological retinal findings should undergo a detailed ophthalmic examination as soon as possible after birth to prevent total retinal detachment, even without a diagnosis of specific inherited retinal vascular diseases. Further investigations performed in collaboration with an international network may reveal more candidate gene variants possibly related to retinopathy of prematurity-like ophthalmological findings such as extraretinal fibrovascular proliferation. [ABSTRACT FROM AUTHOR]

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies." [Display omitted] We identified homozygous loss-of-function variants in ESAM , encoding an endothelial cell adhesion molecule, in thirteen individuals (including four fetuses) showing intracerebral hemorrhage in prenatal age and neurodevelopmental disorders. Our findings contribute to expand an emerging group of diseases caused by alterations of tight-junction genes (e.g., JAM2 , JAM3 , OCLN). [ABSTRACT FROM AUTHOR]

Endothelial cell-selective adhesion molecule (ESAM) is a member of tight junction molecules, highly abundant in the heart and the lung, and plays a role in regulating endothelial cell permeability. We previously reported that mice with genetic ESAM deficiency ( ESAM -/- ) exhibit coronary microvascular dysfunction leading to the development of left ventricular diastolic dysfunction. Here, we hypothesize that ESAM -/- mice display impairments in the pulmonary vasculature, affecting the overall pulmonary vascular resistance (PVR). We utilized ESAM -/- mice and employed isolated, ventilated, and perfused whole lung preparation to assess PVR independently of cardiac function. PVR was assessed in response to stepwise increases in flow, and also in response to perfusion of the endothelium-dependent agonist, bradykinin, the thromboxane analog, U46619, and the nitric oxide (NO) donor sodium nitroprusside (SNP). We found that PVR, at every applied flow rate, is significantly elevated in ESAM -/- mice compared with WT mice. Bradykinin-induced reduction in PVR and U46619-induced increase in PVR were both diminished in ESAM mice exhibited a reduced level of phospho-Ser473-Akt and phospho-Ser1177-eNOS. Furthermore, in human lung microvascular endothelial cells cultured under flow conditions, we found that siRNA-mediated knockdown of ESAM impaired fluid shear stress-induced endothelial cell alignment. Thus, we suggest that ESAM plays an important role in the endothelium-dependent, flow/shear stress- and vasoactive agonist-stimulated, and NO-mediated maintenance of PVR in mice. -/- Our study reveals a novel role for ESAM in contributing to the maintenance of pulmonary vascular resistance under normal physiological conditions. Employing mice with global genetic deficiency of ESAM and using isolated whole lung preparation, we show significant impairments in nitric oxide-mediated pulmonary artery function. In vitro cell culture studies demonstrate impaired fluid shear stress-induced cell alignment in human lung endothelial cells after siRNA-mediated ESAM knockdown.ESAM -/- mice. Inhibition of NO synthase with N (ω)-nitro-l-arginine methyl ester increased agonist-induced PVR in WT but not in ESAM -/- mice. Pulmonary arteries isolated from ESAM -/- mice exhibited a reduced level of phospho-Ser473-Akt and phospho-Ser1177-eNOS. Furthermore, in human lung microvascular endothelial cells cultured under flow conditions, we found that siRNA-mediated knockdown of ESAM impaired fluid shear stress-induced endothelial cell alignment. Thus, we suggest that ESAM plays an important role in the endothelium-dependent, flow/shear stress- and vasoactive agonist-stimulated, and NO-mediated maintenance of PVR in mice. NEW & NOTEWORTHY Our study reveals a novel role for ESAM in contributing to the maintenance of pulmonary vascular resistance under normal physiological conditions. Employing mice with global genetic deficiency of ESAM and using isolated whole lung preparation, we show significant impairments in nitric oxide-mediated pulmonary artery function. In vitro cell culture studies demonstrate impaired fluid shear stress-induced cell alignment in human lung endothelial cells after siRNA-mediated ESAM knockdown.

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes. [ABSTRACT FROM AUTHOR]

Members of the CAR group of Ig‐like type I transmembrane proteins mediate homotypic cell adhesion, share a common overall extracellular domain structure and are closely related at the amino acid sequence level. CAR proteins are often found at tight junctions and interact with intracellular scaffolding proteins, suggesting that they might modulate tight junction assembly or function. However, impairment of tight junction integrity has not been reported in mouse knockout models or zebrafish mutants of CAR members. In contrast, in the same knockout models deficits in gap junction communication were detected in several organ systems, including the atrioventricular node of the heart, smooth muscle cells of the intestine and the ureter and in Sertoli cells of the testes. Possible interactions between BT‐IgSF and connexin41.8 on the disturbed pattern of pigment stripes found in zebrafish mutants and between ESAM and connexin43 during hematopoiesis in the mouse are also discussed. On the basis of the combined data and phenotypic similarities between CAR member mutants and connexin mutants I hypothesize that they primarily play a role in the organization of gap junction communication. Also see the video abstract here: https://youtu.be/i0yq2KhuDAE. [ABSTRACT FROM AUTHOR]

Purpose: Adiponectin an adipokine, produced by mature adipocyte, has an important effect on several aspects of endothelial function, including leukocyte adhesion (mediated by adhesion molecules like intercellular adhesion molecule 1 (ICAM1) endothelial cell selective adhesion molecule ESAM). Recently, it has been linked to vascular endothelial growth factor (VEGF)-modulated angiogenesis. ESAM might also be involved in modulating VEGF-dependent actions. We studied relationship of adiponectin to ESAM, ICAM1, and VEGF in type 2 diabetic patients (T2DP) with or without microvascular complications.Methods: Incident T2DP referred for nephrologic evaluation were included (patients with no nephropathy or stage 1-4 nephropathy). T2DP with stage 5 chronic kidney disease (CKD) were selected from a dialysis center. Clinical, standard laboratory assessment and adiponectin, ESAM, ICAM1, and VEGF (ELISA) were recorded.Results: Eighty-seven patients were included, 15 had no CKD, 30 with stage 1 or 2 CKD, 20 with stage 3 or 4 CKD and 22 patients on dialysis. ESAM was higher in patients with CKD than in those without CKD (p = 0.02), adiponectin, ICAM1, and VEGF were similar. Adiponectin correlated in univariate analysis to ESAM (r = 0.32, p = 0.002), ICAM1 (r = 0.23, p = 0.038), and CRP (r = 0.31, p = 0.012), and inversely to serum albumin (r = − 0.57, < 0.0001). In predialysis patients, adiponectin also correlated to albuminuria (r = 0.54, p < 0.0001) and glomerular filtration rate (r = − 0.46, p = 0.0001). In multivariate regression ESAM (p = 0.04), VEGF (p = 0.03), and albumin (p < 0.0001) are significant predictors of adiponectin. None of these cytokines were different when comparing patients with and without retinopathy.Conclusion: Adiponectin is directly linked to adhesion molecules and VEGF in T2DP. [ABSTRACT FROM AUTHOR]

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and re-establish endothelial contacts, while allowing cell motility. We have compared the contribution of two endothelial cells-pecific adhesion proteins, VE-cadherin (VE-cad/Cdh5) and Esama (endothelial cell-selective adhesion molecule a), during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appears similar to wild type, whereas esama−/−; ve-cad+/− and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, and display intrajunctional gaps, dislocalization of Zo-1 and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis, and both adhesion proteins are central to endothelial cell recognition during anastomosis. [ABSTRACT FROM AUTHOR]

Abstract: In Australian export-registered abattoirs microbiological monitoring is carried out within the E. coli and Salmonella Monitoring (ESAM) program. During the calendar year 2003, the ESAM database indicated a national prevalence of Escherichia coli of around 3.0% for steers/heifers and 7.1% for cows/bulls. An investigation was carried out to attempt to elucidate why some establishments had E. coli prevalence markedly higher or markedly lower than the national average. The investigation was based on a questionnaire completed by fifteen export establishments which provided data on livestock, processing, operator training and management. The responses were verified by site visits and then evaluated for their relationship with ESAM data on E. coli in two stages. In stage 1, E. coli prevalence for each abattoir was plotted against each variable recorded by the questionnaire; no single variable was a reasonable predictor for prevalence of E. coli on carcases. In stage 2, variables influencing contamination were grouped under two categories: contamination on incoming livestock (Problem variables) together with the ability of the plant''s process to deal with such contamination (Process variables). The analysis prompted two main conclusions. Firstly, plants with a large incoming problem with livestock (long haul, high tag score and proportion of cows/bulls slaughtered) plus “poor” processes had higher than average E. coli prevalence. Secondly, plants with hot water decontamination systems had low E. coli prevalence even when there was a substantial incoming problem with livestock, such as a relatively high proportion of cows/bulls. [Copyright &y& Elsevier]

Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell–cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions. [Copyright &y& Elsevier]

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system. [Copyright &y& Elsevier]

Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45 low c-Kit high cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit + cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation., (Copyright © 2020 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)