JAM-C Is Important for Lens Epithelial Cell Proliferation and Lens Fiber Maturation in Murine Lens Development.

Purpose: The underlying mechanism of congenital cataracts caused by deficiency or mutation of junctional adhesion molecule C (JAM-C) gene remains unclear. Our study aims to elucidate the abnormal developmental process in Jamc-/- lenses and reveal the genes related to lens development that JAM-C may regulate. Methods: Jamc knockout (Jamc-/-) mouse embryos and pups were generated for in vivo studies. Four key developmental stages from embryonic day (E) 12.5 to postnatal day (P) 0.5 were selected for the following experiments. Hematoxylin and eosin staining was used for histological analysis. The 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and TUNEL staining were performed to label lens epithelial cell (LEC) proliferation and apoptosis, respectively. Immunofluorescence and Western blot were used to analyze the markers of lens epithelium, cell cycle exit, and lens fiber differentiation. Results: JAM-C was expressed throughout the process of lens development. Deletion of Jamc resulted in decreased lens size and disorganized lens fibers, which arose from E16.5 and aggravated gradually. The LECs of Jamc-/- lenses showed decreased quantity and proliferation, accompanied with reduction of key transcription factor, FOXE3. The fibers in Jamc-/- lenses were disorganized. Moreover, Jamc-deficient lens fibers showed significantly altered distribution patterns of Cx46 and Cx50. The marker of fiber homeostasis, γ-crystallin, was also decreased in the inner cortex and core fibers of Jamc-/- lenses. Conclusions: Deletion of JAM-C exhibits malfunction of LEC proliferation and fiber maturation during murine lens development, which may be related to the downregulation of FOXE3 expression and abnormal localization patterns of Cx46 and Cx50.

Endometriosis and epithelial ovarian cancer: a two-sample Mendelian randomization analysis.

Endometriosis, a prevalent condition, has long been recognized as a chronic and debilitating ailment affecting an estimated 1790 million women worldwide. Observational studies have established a correlation between endometriosis and ovarian cancer. Thus, we endeavored to employ Two-Sample Mendelian Randomization, utilizing summary statistics from a Genome-Wide Association Study of endometriosis and epithelial ovarian cancer, with genetic markers serving as proxies for epithelial ovarian cancer. The analysis revealed a significant correlation between these entities, with an odds ratio (OR) of 1.23 (95% CI 1.11-1.36). Upon histotype-specific examination, robust evidence emerged for an association of endometriosis with the risk of endometrioid carcinoma (OR 1.49, 95% CI 1.24-1.81), clear cell carcinoma (OR 2.56, 95% CI 1.75-3.73), and low malignant potential tumors (OR 1.28, 95% CI 1.08-1.53). These findings provide a theoretical framework for prospective investigations aimed at enhancing the potential therapeutic efficacy of managing endometriosis in averting the onset and progression of ovarian cancer.

LensAge index as a deep learning-based biological age for self-monitoring the risks of age-related diseases and mortality.

Age is closely related to human health and disease risks. However, chronologically defined age often disagrees with biological age, primarily due to genetic and environmental variables. Identifying effective indicators for biological age in clinical practice and self-monitoring is important but currently lacking. The human lens accumulates age-related changes that are amenable to rapid and objective assessment. Here, using lens photographs from 20 to 96-year-olds, we develop LensAge to reflect lens aging via deep learning. LensAge is closely correlated with chronological age of relatively healthy individuals (R2 > 0.80, mean absolute errors of 4.25 to 4.82 years). Among the general population, we calculate the LensAge index by contrasting LensAge and chronological age to reflect the aging rate relative to peers. The LensAge index effectively reveals the risks of age-related eye and systemic disease occurrence, as well as all-cause mortality. It outperforms chronological age in reflecting age-related disease risks (p < 0.001). More importantly, our models can conveniently work based on smartphone photographs, suggesting suitability for routine self-examination of aging status. Overall, our study demonstrates that the LensAge index may serve as an ideal quantitative indicator for clinically assessing and self-monitoring biological age in humans.

A Comparison of Cell-Cell Interaction Prediction Tools Based on scRNA-seq Data.

Computational prediction of cell-cell interactions (CCIs) is becoming increasingly important for understanding disease development and progression. We present a benchmark study of available CCI prediction tools based on single-cell RNA sequencing (scRNA-seq) data. By comparing prediction outputs with a manually curated gold standard for idiopathic pulmonary fibrosis (IPF), we evaluated prediction performance and processing time of several CCI prediction tools, including CCInx, CellChat, CellPhoneDB, iTALK, NATMI, scMLnet, SingleCellSignalR, and an ensemble of tools. According to our results, CellPhoneDB and NATMI are the best performer CCI prediction tools, among the ones analyzed, when we define a CCI as a source-target-ligand-receptor tetrad. In addition, we recommend specific tools according to different types of research projects and discuss the possible future paths in the field.

PDGF-C promotes cell proliferation partially via downregulating BOP1.

Platelet-derived growth factor C (PDGF-C) is a member of PDGF/VEGF family, which is well-known for important functions in the vascular system. It is widely reported that PDGF-C is able to modulate cell proliferation. However, it is still not very clear about this cell modulating mechanism at the molecular level. In a screening of factors regulated by PDGF-C protein, we fished out a factor called block of proliferation 1 (BOP1), which is a pivotal regulator of ribosome biogenesis and cell proliferation. In this study, we investigated the regulation of BOP1 by PDGF-C and its role in modulating cell proliferation. We found that BOP1 was downregulated at both mRNA and protein levels in cells treated with PDGF-C-containing conditioned medium. On the other hand, BOP1 was upregulated in PDGF-C deficient mice. Furthermore, we confirmed that overexpression of BOP1 inhibited HEK293A cell proliferation, whereas knockdown of BOP1 promoted cell proliferation. The mitogenic effect of PDGF-C could be attenuated by downregulation of BOP1. Our results demonstrate a clear PDGF-C-BOP1 signaling that modulates cell proliferation.

VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway.

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption.

BACKGROUND: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood. METHODS: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries. RESULTS: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) activation of the small GTPase RAC1 (Rac family small GTPase 1), thereby restricting RhoA (Ras homolog family member A)/ROCK-mediated cytoskeleton contraction. CONCLUSIONS: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.

PDGF-D-induced immunoproteasome activation and cell-cell interactions.

Platelet-derived growth factor-D (PDGF-D) is abundantly expressed in ocular diseases. Yet, it remains unknown whether and how PDGF-D affects ocular cells or cell-cell interactions in the eye. In this study, using single-cell RNA sequencing (scRNA-seq) and a mouse model of PDGF-D overexpression in retinal pigment epithelial (RPE) cells, we found that PDGF-D overexpression markedly upregulated the key immunoproteasome genes, leading to increased antigen processing/presentation capacity of RPE cells. Also, more than 6.5-fold ligand-receptor pairs were found in the PDGF-D overexpressing RPE-choroid tissues, suggesting markedly increased cell-cell interactions. Moreover, in the PDGF-D-overexpressing tissues, a unique cell population with a transcriptomic profile of both stromal cells and antigen-presenting RPE cells was detected, suggesting PDGF-D-induced epithelial-mesenchymal transition of RPE cells. Importantly, administration of ONX-0914, an immunoproteasome inhibitor, suppressed choroidal neovascularization (CNV) in a mouse CNV model in vivo. Together, we show that overexpression of PDGF-D increased pro-angiogenic immunoproteasome activities, and inhibiting immunoproteasome pathway may have therapeutic value for the treatment of neovascular diseases.

Identification of the regulatory mechanism of ACE2 in COVID-19-induced kidney damage with systems genetics approach.

Studies showed that SARS-CoV-2 can directly target the kidney and induce renal damage. As the cell surface receptor for SARS-CoV-2 infection, the angiotensin-converting enzyme 2 (ACE2) plays a pivotal role for renal physiology and function. Thus, it is important to understand ACE2 through which pathway influences the pathogenesis of renal damage induced by COVID-19. In this study, we first performed an eQTL mapping for Ace2 in kidney tissues in 53 BXD mice strains. Results demonstrated that Ace2 is highly expressed and strongly controlled by a genetic locus on chromosome 16 in the kidney, with six genes (Dnase1, Vasn, Usp7, Abat, Mgrn1, and Rbfox1) dominated as the upstream modulator, as they are highly correlated with Ace2 expression. Gene co-expression analysis showed that Ace2 co-variates are significantly involved in the renin-angiotensin system (RAS) pathway which acts as a reno-protector. Importantly, we also found that Ace2 is positively correlated with Pdgf family members, particularly Pdgfc, which showed the most association among the 76 investigated growth factors. Mammalian Phenotype Ontology enrichment indicated that the cognate transcripts for both Ace2 and Pdgfc were mainly involved in regulating renal physiology and morphology. Among which, Cd44, Egfr, Met, Smad3, and Stat3 were identified as hub genes through protein-protein interaction analysis. Finally, in aligning with our systems genetics findings, we found ACE2, pdgf family members, and RAS genes decreased significantly in the CAKI-1 kidney cancer cells treated with S protein and receptor binding domain structural protein. Collectively, our data suggested that ACE2 work with RAS, PDGFC, as well as their cognate hub genes to regulate renal function, which could guide for future clinical prevention and targeted treatment for COVID-19-induced renal damage outcomes. KEY MESSAGES: • Ace2 is highly expressed and strongly controlled by a genetic locus on chromosome 16 in the kidney. • Ace2 co-variates are enriched in the RAS pathway. • Ace2 is strongly correlated with the growth factor Pdgfc. • Ace2 and Pdgfc co-expressed genes involved in the regulation of renal physiology and morphology. • SARS-CoV-2 spike glycoprotein induces down-regulation of Ace2, RAS, and Pdgfc.

Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis.

Although mitogen-inducible gene 6 (MIG6) is highly expressed in vascular endothelial cells, it remains unknown whether MIG6 affects vascular permeability. Here, we show for the first time a critical role of MIG6 in limiting vascular permeability. We unveil that genetic deletion of Mig6 in mice markedly increased VEGFA-induced vascular permeability, and MIG6 knockdown impaired endothelial barrier function. Mechanistically, we reveal that MIG6 inhibits VEGFR2 phosphorylation by binding to the VEGFR2 kinase domain 2, and MIG6 knockdown increases the downstream signaling of VEGFR2 by enhancing phosphorylation of PLCγ1 and eNOS. Moreover, MIG6 knockdown disrupted the balance between RAC1 and RHOA GTPase activation, leading to endothelial cell barrier breakdown and the elevation of vascular permeability. Our findings demonstrate an essential role of MIG6 in maintaining endothelial cell barrier integrity and point to potential therapeutic implications of MIG6 in the treatment of diseases involving vascular permeability. Xing et al. (2022) investigated the critical role of MIG6 in vascular permeability. MIG6 deficiency promotes VEGFA-induced vascular permeability via activation of PLCγ1-Ca2+-eNOS signaling and perturbation of the balance in RAC1/RHOA activation, resulting in endothelial barrier disruption.

Deficiency of Jamc Leads to Congenital Nuclear Cataract and Activates the Unfolded Protein Response in Mouse Lenses.

Purpose: The malfunction of junctional adhesion molecule C (JAM-C) has been reported to induce congenital cataract in humans and mice; however, specific characters and the mechanism of this cataract are still unclear. This study aimed to characterize abnormal lens development in Jamc knockout mice and clarify the underlying mechanism. Methods: Jamc knockout mice backcrossed onto the C57BL/6 genetic background were used for this research. Slit-lamp and darkfield images showed the cataract phenotype of Jamc-/- mice. Hematoxylin and eosin staining was performed to visualize the morphological and histological features. RNA sequencing was applied to detect differentially expressed genes. Quantitative RT-PCR, western blot, and immunofluorescence were used to determine the level of unfolded protein response (UPR)-related genes. TUNEL staining was utilized to label cell death. Results: Jamc knockout mice exhibited nuclear cataract with abnormal lens morphology and defective degradation of nuclei and organelles in lens fiber cells. Compared with wild-type control mice, the expression level of BiP, CHOP, TRIB3, and CHAC1, genes involved in endoplasmic reticulum stress and the UPR, were highly upregulated in Jamc-/- lenses, suggesting that abnormal lens development was accompanied by UPR activation. Moreover, increased cell death was also found in Jamc-/- lenses. Conclusions: Congenital nuclear cataract caused by Jamc deficiency is accompanied by defective degradation of nuclei and organelles in lens fiber cells, lens structure disorder, and UPR activation, suggesting that JAM-C is required for maintaining normal lens development and that UPR activation is involved in cataract formation in Jamc-deficient lenses.

Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast.

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-γ and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-γ signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-γ link for BC treatment.

GAD1 alleviates injury-induced optic neurodegeneration by inhibiting retinal ganglion cell apoptosis.

The degeneration of the optic nerve narrows the visual field, eventually causing overall vision loss. This study aimed to identify global protein changes in the retina of optic nerve crushing (ONC) mice and to identify key regulators and pathways involved in injury-induced cell death during the progression of optic neurodegeneration. Label-free quantitative proteomics combined with bioinformatic analysis was performed on retinal protein extracts from ONC and sham-operated mice. Among the 1433 proteins detected, 121 proteins were differentially expressed in the retina of ONC mice. Further bioinformatic analysis showed that various metabolic pathways, including glutamate metabolism and γ-aminobutyric acid (GABA) synthesis, were significantly dysregulated in the injured mouse retinas. Glutamate decarboxylase 1 (GAD1) is the enzyme that converts glutamate into GABA, which was significantly up-regulated during ONC injury. Exogenous GAD1 treatment increased retinal ganglion cell (RGC) survival in the ONC-injured retina. In addition, changes in GAD1 expression were also observed in several other ophthalmic diseases. Vascular endothelial growth factor B (VEGF-B) has previously been reported to protect RGCs from apoptosis and positively regulated the expression of GAD1 in the retina. Notably, combination treatment with GAD1 and VEGF-B also provided strong protection against injury-induced RGC apoptosis. These results suggest that GAD1 expression may serve as an intrinsic protective mechanism that is commonly activated during retinal injury. Targeting GAD1 may serve as a potential strategy to treat optic neurodegenerative diseases.

Chromatin Hubs: A biological and computational outlook.

This review discusses our current understanding of chromatin biology and bioinformatics under the unifying concept of "chromatin hubs." The first part reviews the biology of chromatin hubs, including chromatin-chromatin interaction hubs, chromatin hubs at the nuclear periphery, hubs around macromolecules such as RNA polymerase or lncRNAs, and hubs around nuclear bodies such as the nucleolus or nuclear speckles. The second part reviews existing computational methods, including enhancer-promoter interaction prediction, network analysis, chromatin domain callers, transcription factory predictors, and multi-way interaction analysis. We introduce an integrated model that makes sense of the existing evidence. Understanding chromatin hubs may allow us (i) to explain long-unsolved biological questions such as interaction specificity and redundancy of mechanisms, (ii) to develop more realistic kinetic and functional predictions, and (iii) to explain the etiology of genomic disease.

PDGFD switches on stem cell endothelial commitment.

The critical factors regulating stem cell endothelial commitment and renewal remain not well understood. Here, using loss- and gain-of-function assays together with bioinformatic analysis and multiple model systems, we show that PDGFD is an essential factor that switches on endothelial commitment of embryonic stem cells (ESCs). PDGFD genetic deletion or knockdown inhibits ESC differentiation into EC lineage and increases ESC self-renewal, and PDGFD overexpression activates ESC differentiation towards ECs. RNA sequencing reveals a critical requirement of PDGFD for the expression of vascular-differentiation related genes in ESCs. Importantly, PDGFD genetic deletion or knockdown increases ESC self-renewal and decreases blood vessel densities in both embryonic and neonatal mice and in teratomas. Mechanistically, we reveal that PDGFD fulfills this function via the MAPK/ERK pathway. Our findings provide new insight of PDGFD as a novel regulator of ESC fate determination, and suggest therapeutic implications of modulating PDGFD activity in stem cell therapy.

Regional Downregulation of Dopamine Receptor D1 in Bilateral Dorsal Lateral Geniculate Nucleus of Monocular Form-Deprived Amblyopia Models.

Amblyopia is a common eye disease characterized by impaired best-corrected visual acuity. It starts in early childhood and leads to permanent vision reduction if left untreated. Even though many young patients with amblyopia are well treated in clinical practice, the underlying mechanism remains to be elucidated, which limits not only our understanding of this disease but also the therapeutic approach. To investigate the molecular mechanism of amblyopia, primate and rodent models of monocular-deprived amblyopia were created for mRNA screening and confirmation. We obtained 818 differentially expressed genes from the dorsal lateral geniculate nucleus (dLGN) of a primate model of amblyopia. After Gene Ontology and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses, the main enriched pathways were related to neural development. Interestingly, a particular neurotransmitter pathway, the dopaminergic pathway, was identified. The downregulation of dopamine receptor D1 (DRD1) was confirmed in both monkey and mouse samples. Furthermore, the immunofluorescence staining indicated that DRD1 expression was downregulated in both ventrolateral region of the contralateral dLGN and the dorsomedial region of the ipsilateral dLGN in the mouse model. The regions with downregulated expression of DRD1 were the downstream targets of the visual projection from the amblyopic eye. This study suggested that the downregulation of DRD1 in the LGN may be a cause for amblyopia. This may also be a reason for the failure of some clinical cases of levodopa combined with carbidopa applied to amblyopes.

Corrigendum: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization.

[This corrects the article DOI: 10.3389/fcell.2021.686886.].

Expression and function of PDGF-C in development and stem cells.

Platelet-derived growth factor C (PDGF-C) is a relatively new member of the PDGF family, discovered nearly 20 years after the finding of platelet-derived growth factor A (PDGF-A) and platelet-derived growth factor B (PDGF-B). PDGF-C is generally expressed in most organs and cell types. Studies from the past 20 years have demonstrated critical roles of PDGF-C in numerous biological, physiological and pathological processes, such as development, angiogenesis, tumour growth, tissue remodelling, wound healing, atherosclerosis, fibrosis, stem/progenitor cell regulation and metabolism. Understanding PDGF-C expression and activities thus will be of great importance to various research disciplines. In this review, however, we mainly discuss the expression and functions of PDGF-C and its receptors in development and stem cells.

Automatic cell type identification methods for single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for scientists of many research disciplines due to its ability to elucidate the heterogeneous and complex cell-type compositions of different tissues and cell populations. Traditional cell-type identification methods for scRNA-seq data analysis are time-consuming and knowledge-dependent for manual annotation. By contrast, automatic cell-type identification methods may have the advantages of being fast, accurate, and more user friendly. Here, we discuss and evaluate thirty-two published automatic methods for scRNA-seq data analysis in terms of their prediction accuracy, F1-score, unlabeling rate and running time. We highlight the advantages and disadvantages of these methods and provide recommendations of method choice depending on the available information. The challenges and future applications of these automatic methods are further discussed. In addition, we provide a free scRNA-seq data analysis package encompassing the discussed automatic methods to help the easy usage of them in real-world applications.