Connexins are the structural components of gap junctions and hemichannels that mediate the communication and exchange of small molecules between cells, and between the intracellular and extracellular environment, respectively. Connexin (Cx) 46 is predominately expressed in lens fiber cells, where they function in maintaining the homeostasis and transparency of the lens. Cx46 mutations are associated with impairment of channel function, which results in the development of congenital cataracts. Cx46 gap junctions and hemichannels are closely regulated by multiple mechanisms. Key regulators of Cx46 channel function include Ca²⁺ and calmodulin (CaM). Ca²⁺ plays an essential role in lens homeostasis, and its dysregulation causes cataracts. Ca²⁺ associated CaM is a well-established inhibitor of gap junction coupling. Recent studies suggest that elevated intracellular Ca²⁺ activates Cx hemichannels in lens fiber cells and Cx46 directly interacts with CaM. A Cx46 site mutation (Cx46-G143R), which is associated with congenital Coppock cataracts, shows an increased Cx46-CaM interaction and this interaction is insensitive to Ca²⁺, given that depletion of Ca²⁺ reduces the interaction between CaM and wild-type Cx46. Moreover, inhibition of CaM function greatly reduces the hemichannel activity in the Cx46 G143R mutant. These research findings suggest a new regulatory mechanism by which enhanced association of Cx46 with CaM leads to the increase in hemichannel activity and dysregulation may lead to cataract development. In this review, we will first discuss the involvement of Ca²⁺/CaM in lens homeostasis and pathology, and follow by providing a general overview of Ca²⁺/CaM in the regulation of Cx46 gap junctions. We discuss the most recent studies concerning the molecular mechanism of Ca²⁺/CaM in regulating Cx46 hemichannels. Finally, we will offer perspectives of the impacts of Ca²⁺/CaM and dysregulation on Cx46 channels and vice versa.

BACKGROUND: EMCN (endomucin), 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 VEGF (vascular endothelial growth factor) activity through modulating VEGFR2 (VEGF receptor 2) 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. METHODS: Global EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was used to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, whereas urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP (enhanced green fluorescent protein), CD45 (cluster of differentiation 45), CD31, CD34, podocin, and albumin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a noninvasive 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. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. RESULTS: The EMCN ^−/− mice exhibited increased infiltration of CD45 ⁺ cells, with an increased proportion of Ly6G ^high Ly6C ^high myeloid cells and higher VCAM-1 (vascular cell adhesion molecule 1) expression. EMCN ^−/− mice displayed albuminuria with increased albumin in the Bowman’s space compared with 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 (mitogen-activated protein kinase)/ERK (extracellular signal–regulated kinase) 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. CONCLUSION: 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.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.

To investigate the long-term outcomes and complications of scleral-fixated intraocular lens (SFIOL) implantation without conjunctival peritomies and sclerotomy in patients with a history of ocular trauma with inadequate capsular support during primary pars plana vitrectomy or silicone oil removal. Records of ocular trauma patients who underwent implantation of SFIOL without conjunctival peritomies and sclerotomy during primary pars plana vitrectomy or silicone oil removal. Sixty-nine eyes of 69 patients were included in this study. The median follow-up period was 34 months (range, 6–99 months). The average patient age at the time of surgery was 44 years old (range, 4–80 years). At the end of follow-up, the preoperative mean of best corrected visual acuity (BCVA) was 0.79 ± 0.86 log of the minimum angle of resolution (logMAR), which improved 0.20 ± 0.26 logMAR postoperatively (P = 0.01). BCVA improved or remained unchanged in 64 eyes (92.8%), VA decreased two lines in five eyes (7.2%). Early postoperative complications included transient corneal edema in seven eyes (10.1%), minor vitreous hemorrhage in four eyes (5.8%), transient elevated intraocular pressure (IOP) in one eye (1.4%), and hypotony in three eyes (4.3%). Late postoperative complications included persistent elevated IOP in five eyes (7.2%), epiretinal membrane formation in three eyes (4.3%), and cystoid macular edema noted in one eye (1.4%). Use of a scleral-fixated intraocular lens implantation without conjunctival peritomies and sclerotomy in ocular trauma patients during either primary pars plana vitrectomy or second silicone oil removal is a valuable approach for the management of traumatic aphakia in the absence of capsular support.

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN’s involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization. Graphical Abstract