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Deletion of endothelial glycocalyx component endomucin impairs retinal vascularization and suppresses pathological neovascularization

ABSTRACT: Endomucin (EMCN), an endothelial-selective glycocalyx component highly expressed in capillaries and venous endothelium, plays a critical role in regulating vascular endothelial growth factor receptor 2 (VEGFR2) endocytosis and downstream of VEGF signaling. Using a global EMCN knockout mouse model, we investigated the effects of EMCN deficiency on retinal vascularization during development and pathological angiogenesis. While Emcn-/- mice showed no significant difference in the avascular area in an oxygen-induced retinopathy (OIR) model, there was a significant reduction in neovascular tufts at postnatal day 17. RNA seq of the OIR retinas with gene ontology enrichment analysis indicate response to hypoxia and angiogenesis pathways were differentially regulated in Emcn-/- mice in OIR.

ORGANISM(S): Mus musculus

PROVIDER: GSE288833 | GEO | 2026/05/15

REPOSITORIES: GEO

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Project description: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 sequencing. 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 Ly6GhighLy6Chigh 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 sequencing 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.

Project description:Aims: Novel cancer therapies leading to increased survivorship of cancer patients have been negated by a concomitant rise in cancer therapies-related cardiovascular toxicities. Sunitinib, a first line multi receptor tyrosine kinase inhibitor (TKI), has been reported to cause vascular dysfunction although the initiating mechanisms contributing to this side effect remain unknown. Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in endothelial cells (ECs); however, their roles in cancer therapies-related vascular toxicities remain underexplored. Methods and Results: We performed lncRNA expression profiling to identify potential lncRNAs that are dysregulated in human induced pluripotent stem cells-derived ECs (iPSC-ECs) treated with sunitinib. We show that the lncRNA hyaluronan synthase 2 antisense 1 (HAS2-AS1) is significantly diminished in sunitinib-treated iPSC-ECs. Sunitinib was found to downregulate HAS2-AS1 by an epigenetic mechanism involving hypermethylation. Depletion of HAS2-AS1 recapitulated sunitinib-induced detrimental effects on iPSC-ECs, whereas CRISPR-mediated activation of HAS2-AS1 reversed sunitinib-induced dysfunction. We confirm that HAS2-AS1 stabilizes the expression of its sense gene HAS2 via an RNA/mRNA heteroduplex formation. Knockdown of HAS2-AS1 led to reduced synthesis of hyaluronic acid (HA) and upregulation of ADAMTS5, an enzyme involved in extracellular matrix degradation, resulting in disruption of the endothelial glycocalyx which is critical for ECs. In vivo, sunitinib-treated mice showed reduced coronary flow reserve, accompanied by a reduction in has2os and degradation of the endothelial glycocalyx. Finally, we identify that treatment with high molecular-weight HA can prevent the deleterious effects of sunitinib both in vitro and in vivo by preserving the endothelial glycocalyx. Conclusions: Our findings highlight the importance of lncRNA-mediated regulation of the endothelial glycocalyx as an important determinant of sunitinib-induced vascular toxicity and reveal potential novel therapeutic avenues to attenuate sunitinib-induced vascular dysfunction.

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Deletion of endothelial glycocalyx component endomucin impairs retinal vascularization and suppresses pathological neovascularization

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