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1.
Nat Commun ; 14(1): 7300, 2023 11 11.
Article in English | MEDLINE | ID: mdl-37949852

ABSTRACT

Anterior Uveitis (AU) is the inflammation of the anterior part of the eye, the iris and ciliary body and is strongly associated with HLA-B*27. We report AU exome sequencing results from eight independent cohorts consisting of 3,850 cases and 916,549 controls. We identify common genome-wide significant loci in HLA-B (OR = 3.37, p = 1.03e-196) and ERAP1 (OR = 0.86, p = 1.1e-08), and find IPMK (OR = 9.4, p = 4.42e-09) and IDO2 (OR = 3.61, p = 6.16e-08) as genome-wide significant genes based on the burden of rare coding variants. Dividing the cohort into HLA-B*27 positive and negative individuals, we find ERAP1 haplotype is strongly protective only for B*27-positive AU (OR = 0.73, p = 5.2e-10). Investigation of B*27-negative AU identifies a common signal near HLA-DPB1 (rs3117230, OR = 1.26, p = 2.7e-08), risk genes IPMK and IDO2, and several additional candidate risk genes, including ADGFR5, STXBP2, and ACHE. Taken together, we decipher the genetics underlying B*27-positive and -negative AU and identify rare and common genetic signals for both subtypes of disease.


Subject(s)
Uveitis, Anterior , Humans , Uveitis, Anterior/genetics , Inflammation/genetics , Haplotypes , Genes, MHC Class I , HLA-B Antigens/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Aminopeptidases/genetics , Minor Histocompatibility Antigens
2.
Curr Oncol ; 30(9): 8586-8601, 2023 09 19.
Article in English | MEDLINE | ID: mdl-37754538

ABSTRACT

Pediatric Brain Tumor Survivors (PBTS) often experience social, academic and employment difficulties during aftercare. Despite their needs, they often do not use the services available to them. Following a previous qualitative study, we formulated solutions to help support PBTS return to daily activities after treatment completion. The present study aims to confirm and prioritize these solutions with a larger sample. We used a mixed-methods survey with 68 participants (43 survivors, 25 parents, PBTS' age: 15-39 years). Firstly, we collected information about health condition, and school/work experience in aftercare. Then, we asked participants to prioritize the previously identified solutions using Likert scales and open-ended questions. We used descriptive and inferential statistics to analyze data, and qualitative information to support participants' responses. Participants prioritized the need for evaluation, counseling, and follow-up by health professionals to better understand their post-treatment needs, obtain help to access adapted services, and receive information about resources at school/work. Responses to open-ended questions highlighted major challenges regarding the implementation of professionals' recommendations at school/work and the need for timely interventions. These results will help refine solutions for PBTS and provide key elements for future implementation. Translating these priorities into action will need further work involving professionals and decision makers.


Subject(s)
Brain Neoplasms , Child , Humans , Adolescent , Young Adult , Adult , Brain Neoplasms/therapy , Employment , Health Personnel , Parents , Survivors
3.
Nat Genet ; 55(8): 1277-1287, 2023 08.
Article in English | MEDLINE | ID: mdl-37558884

ABSTRACT

In this study, we leveraged the combined evidence of rare coding variants and common alleles to identify therapeutic targets for osteoporosis. We undertook a large-scale multiancestry exome-wide association study for estimated bone mineral density, which showed that the burden of rare coding alleles in 19 genes was associated with estimated bone mineral density (P < 3.6 × 10-7). These genes were highly enriched for a set of known causal genes for osteoporosis (65-fold; P = 2.5 × 10-5). Exome-wide significant genes had 96-fold increased odds of being the top ranked effector gene at a given GWAS locus (P = 1.8 × 10-10). By integrating proteomics Mendelian randomization evidence, we prioritized CD109 (cluster of differentiation 109) as a gene for which heterozygous loss of function is associated with higher bone density. CRISPR-Cas9 editing of CD109 in SaOS-2 osteoblast-like cell lines showed that partial CD109 knockdown led to increased mineralization. This study demonstrates that the convergence of common and rare variants, proteomics and CRISPR can highlight new bone biology to guide therapeutic development.


Subject(s)
Genetic Predisposition to Disease , Osteoporosis , Humans , Exome Sequencing , Osteoporosis/genetics , Bone Density/genetics , Alleles , Transcription Factors/genetics , Genome-Wide Association Study
4.
Nat Genet ; 55(7): 1138-1148, 2023 07.
Article in English | MEDLINE | ID: mdl-37308787

ABSTRACT

Human genetic studies of smoking behavior have been thus far largely limited to common variants. Studying rare coding variants has the potential to identify drug targets. We performed an exome-wide association study of smoking phenotypes in up to 749,459 individuals and discovered a protective association in CHRNB2, encoding the ß2 subunit of the α4ß2 nicotine acetylcholine receptor. Rare predicted loss-of-function and likely deleterious missense variants in CHRNB2 in aggregate were associated with a 35% decreased odds for smoking heavily (odds ratio (OR) = 0.65, confidence interval (CI) = 0.56-0.76, P = 1.9 × 10-8). An independent common variant association in the protective direction ( rs2072659 ; OR = 0.96; CI = 0.94-0.98; P = 5.3 × 10-6) was also evident, suggesting an allelic series. Our findings in humans align with decades-old experimental observations in mice that ß2 loss abolishes nicotine-mediated neuronal responses and attenuates nicotine self-administration. Our genetic discovery will inspire future drug designs targeting CHRNB2 in the brain for the treatment of nicotine addiction.


Subject(s)
Nicotine , Tobacco Use Disorder , Humans , Animals , Mice , Smoking/genetics , Tobacco Use Disorder/genetics , Phenotype , Odds Ratio
6.
Nature ; 612(7939): 301-309, 2022 12.
Article in English | MEDLINE | ID: mdl-36450978

ABSTRACT

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.


Subject(s)
COVID-19 , Cardiovascular Diseases , Humans , Clonal Hematopoiesis/genetics , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/genetics
7.
Nat Commun ; 13(1): 4844, 2022 08 23.
Article in English | MEDLINE | ID: mdl-35999217

ABSTRACT

Body fat distribution is a major, heritable risk factor for cardiometabolic disease, independent of overall adiposity. Using exome-sequencing in 618,375 individuals (including 160,058 non-Europeans) from the UK, Sweden and Mexico, we identify 16 genes associated with fat distribution at exome-wide significance. We show 6-fold larger effect for fat-distribution associated rare coding variants compared with fine-mapped common alleles, enrichment for genes expressed in adipose tissue and causal genes for partial lipodystrophies, and evidence of sex-dimorphism. We describe an association with favorable fat distribution (p = 1.8 × 10-09), favorable metabolic profile and protection from type 2 diabetes (~28% lower odds; p = 0.004) for heterozygous protein-truncating mutations in INHBE, which encodes a circulating growth factor of the activin family, highly and specifically expressed in hepatocytes. Our results suggest that inhibin ßE is a liver-expressed negative regulator of adipose storage whose blockade may be beneficial in fat distribution-associated metabolic disease.


Subject(s)
Diabetes Mellitus, Type 2 , Inhibin-beta Subunits/genetics , Adipose Tissue , Adiposity/genetics , Diabetes Mellitus, Type 2/genetics , Exome/genetics , Humans , Mutation
8.
N Engl J Med ; 387(4): 332-344, 2022 07 28.
Article in English | MEDLINE | ID: mdl-35939579

ABSTRACT

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).


Subject(s)
Apoptosis Regulatory Proteins , Germ-Line Mutation , Liver Diseases , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Genetic Predisposition to Disease/genetics , Genetic Predisposition to Disease/prevention & control , Humans , Liver/metabolism , Liver Diseases/genetics , Liver Diseases/metabolism , Liver Diseases/prevention & control , Transaminases/genetics , Exome Sequencing
9.
NPJ Genom Med ; 6(1): 73, 2021 Sep 08.
Article in English | MEDLINE | ID: mdl-34497273

ABSTRACT

The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1+KRT5loMUC5AC- club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air-liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology.

10.
PLoS One ; 15(9): e0237529, 2020.
Article in English | MEDLINE | ID: mdl-32941426

ABSTRACT

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.


Subject(s)
Idiopathic Pulmonary Fibrosis/pathology , Lung/pathology , Transcriptome , Bronchioles/cytology , Bronchioles/pathology , Humans , Idiopathic Pulmonary Fibrosis/genetics , Lung/cytology , Respiratory Mucosa/cytology , Respiratory Mucosa/pathology , Secretoglobins/genetics , Single-Cell Analysis , Uteroglobin/genetics
11.
Respir Res ; 21(1): 200, 2020 Jul 29.
Article in English | MEDLINE | ID: mdl-32727470

ABSTRACT

BACKGROUND: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. METHODS: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. RESULTS: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. CONCLUSIONS: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.


Subject(s)
Cigarette Smoking/genetics , Genetic Testing/methods , Lung Diseases/genetics , Respiratory Mucosa/physiology , Sequence Analysis, RNA/methods , Transcriptome/genetics , Airway Remodeling/genetics , Bronchoscopy/methods , Cigarette Smoking/adverse effects , Gene Expression , Humans , Lung Diseases/diagnosis , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/genetics , Respiratory Mucosa/pathology
12.
Cells ; 9(6)2020 06 05.
Article in English | MEDLINE | ID: mdl-32517158

ABSTRACT

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.


Subject(s)
Sialomucins/chemistry , Sialomucins/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Amino Acid Sequence , Endocytosis , Glycosylation , Humans , Mutation/genetics , Protein Domains , Sialomucins/genetics , Signal Transduction
13.
Sci Rep ; 10(1): 6257, 2020 04 10.
Article in English | MEDLINE | ID: mdl-32277131

ABSTRACT

Cigarette smoke (CS) is the leading risk factor to develop COPD. Therefore, the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC) were investigated, using cells from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. RNA sequencing analyses revealed the deregulation of marker genes for basal and secretory cells upon CS exposure. The comparison between gene signatures obtained from the in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. non-smoker) revealed a high degree of similarity between deregulated genes and pathways induced by CS. Taken together, whole cigarette smoke alters the differentiation of small airway basal cells in vitro. The established model showed a good translatability to the situation in vivo. Thus, the model can help to identify and test novel therapeutic approaches to restore the impaired epithelial repair mechanisms in COPD, which is still a high medical need.


Subject(s)
Bronchioles/pathology , Cell Differentiation/drug effects , Epithelial Cells/pathology , Pulmonary Disease, Chronic Obstructive/pathology , Smoke/adverse effects , Tobacco Products/toxicity , Adult , Aged , Bronchioles/cytology , Bronchioles/drug effects , Cells, Cultured , Epithelial Cells/drug effects , Female , Humans , Male , Middle Aged , Primary Cell Culture , Pulmonary Disease, Chronic Obstructive/etiology , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects , Respiratory Mucosa/pathology , Smoking/adverse effects
14.
J Biol Chem ; 295(19): 6641-6651, 2020 05 08.
Article in English | MEDLINE | ID: mdl-32193206

ABSTRACT

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.


Subject(s)
ADAM10 Protein/metabolism , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Membrane Proteins/metabolism , Sialoglycoproteins/metabolism , Tumor Necrosis Factor-alpha/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Humans , Inflammation/metabolism , Inflammation/pathology
15.
FASEB J ; 33(8): 9362-9373, 2019 08.
Article in English | MEDLINE | ID: mdl-31141406

ABSTRACT

We have previously shown that knockdown of endomucin (EMCN), an integral membrane glycocalyx glycoprotein, prevents VEGF-induced proliferation, migration, and tube formation in vitro and angiogenesis in vivo. In the endothelium, VEGF mediates most of its angiogenic effects through VEGF receptor 2 (VEGFR2). To understand the role of EMCN, we examined the effect of EMCN depletion on VEGFR2 endocytosis and activation. Results showed that although VEGF stimulation promoted VEGFR2 internalization in control endothelial cells (ECs), loss of EMCN prevented VEGFR2 endocytosis. Cell surface analysis revealed a decrease in VEGFR2 following VEGF stimulation in control but not siRNA directed against EMCN-transfected ECs. EMCN depletion resulted in heightened phosphorylation following VEGF stimulation with an increase in total VEGFR2 protein. These results indicate that EMCN modulates VEGFR2 endocytosis and activity and point to EMCN as a potential therapeutic target.-LeBlanc, M. E., Saez-Torres, K. L., Cano, I., Hu, Z., Saint-Geniez, M., Ng, Y.-S., D'Amore, P. A. Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.


Subject(s)
Glycocalyx/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Adenoviridae/genetics , Cell Line , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Endocytosis/drug effects , Endocytosis/genetics , Endocytosis/physiology , Humans , Phosphorylation/drug effects , Sialomucins/genetics , Sialomucins/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-2/genetics
16.
Angiogenesis ; 22(3): 369-382, 2019 08.
Article in English | MEDLINE | ID: mdl-30644010

ABSTRACT

Retinopathy of prematurity (ROP) with pathological retinal neovascularization is the most common cause of blindness in children. ROP is currently treated with laser therapy or cryotherapy, both of which may adversely affect the peripheral vision with limited efficacy. Owing to the susceptibility of the developing retina and vasculatures to pharmacological intervention, there is currently no approved drug therapy for ROP in preterm infants. Secretogranin III (Scg3) was recently discovered as a highly disease-restricted angiogenic factor, and a Scg3-neutralizing monoclonal antibody (mAb) was reported with high efficacy to alleviate oxygen-induced retinopathy (OIR) in mice, a surrogate model of ROP. Herein we independently investigated the efficacy of anti-Scg3 mAb in OIR mice and characterized its safety in neonatal mice. We developed a new Scg3-neutralizing mAb recognizing a distinct epitope and independently established the therapeutic activity of anti-Scg3 therapy to alleviate OIR-induced pathological retinal neovascularization in mice. Importantly, anti-Scg3 mAb showed no detectable adverse effects on electroretinography and developing retinal vasculature. Furthermore, systemic anti-Scg3 mAb induced no renal tubular injury or abnormality in kidney vessel development and body weight gain of neonatal mice. In contrast, anti-vascular endothelial growth factor drug aflibercept showed significant side effects in neonatal mice. These results suggest that anti-Scg3 mAb may have the safety and efficacy profiles required for ROP therapy.


Subject(s)
Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/therapeutic use , Chromogranins/antagonists & inhibitors , Retinopathy of Prematurity/drug therapy , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/therapeutic use , Female , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intravitreal Injections , Kidney/drug effects , Kidney/growth & development , Kidney/pathology , Male , Mice, Inbred C57BL , Oxygen , Retinal Vessels/drug effects , Retinal Vessels/pathology , Retinal Vessels/physiopathology , Retinopathy of Prematurity/physiopathology , Weight Gain/drug effects
17.
Exp Eye Res ; 181: 120-126, 2019 04.
Article in English | MEDLINE | ID: mdl-30633921

ABSTRACT

Wet age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. The advent of anti-vascular endothelial growth factor (VEGF) drugs represents a major breakthrough in wet AMD therapy but with limited efficacy to improve visual acuity. Secretogranin III (Scg3, SgIII) was recently discovered as a novel angiogenic factor with VEGF-independent mechanisms. Scg3-neutralizing monoclonal antibody (mAb) was reported to alleviate pathological retinal neovascularization in oxygen-induced retinopathy mice and retinal vascular leakage in diabetic mice with high efficacy and disease selectivity. Herein we investigated whether Scg3 is a novel angiogenic target for CNV therapy in mouse models. We found that anti-Scg3 ML49.3 mAb inhibited Scg3-induced proliferation and Src phosphorylation in human retinal microvascular endothelial cells. Intravitreal injection of Scg3-neutralizing polyclonal antibodies (pAb) or mAb significantly attenuated laser-induced CNV leakage, CNV 3D volume, lesion area and vessel density. Furthermore, subcutaneous administration of Scg3-neutralizing pAb or mAb significantly prevented Matrigel-induced CNV. The efficacy of anti-Scg3 pAb or mAb was comparable to VEGF inhibitor aflibercept. These findings suggest that Scg3 plays an important role in CNV pathogenesis and that anti-Scg3 mAb efficiently ameliorates laser- or Matrigel-induced CNV.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antibodies, Neutralizing/pharmacology , Choroidal Neovascularization/drug therapy , Chromogranins/pharmacology , Endothelial Cells/drug effects , Molecular Targeted Therapy/methods , Analysis of Variance , Animals , Cell Proliferation/drug effects , Disease Models, Animal , Female , Intravitreal Injections , Male , Mice , Mice, Inbred C57BL , Retina/cytology
18.
Cell Mol Life Sci ; 75(4): 635-647, 2018 02.
Article in English | MEDLINE | ID: mdl-28856381

ABSTRACT

Secretogranin III (Scg3) is a member of the granin protein family that regulates the biogenesis of secretory granules. Scg3 was recently discovered as an angiogenic factor, expanding its functional role to extrinsic regulation. Unlike many other known angiogenic factors, the pro-angiogenic actions of Scg3 are restricted to pathological conditions. Among thousands of quantified endothelial ligands, Scg3 has the highest binding activity ratio to diabetic vs. healthy mouse retinas and lowest background binding to normal vessels. In contrast, vascular endothelial growth factor binds to and stimulates angiogenesis of both diabetic and control vasculature. Consistent with its role in pathological angiogenesis, Scg3-neutralizing antibodies alleviate retinal vascular leakage in mouse models of diabetic retinopathy and retinal neovascularization in oxygen-induced retinopathy mice. This review summarizes our current knowledge of Scg3 as a regulatory protein of secretory granules, highlights its new role as a highly disease-selective angiogenic factor, and envisions Scg3 inhibitors as "selective angiogenesis blockers" for targeted therapy.


Subject(s)
Angiogenesis Inducing Agents/metabolism , Chromogranins/physiology , Diabetic Retinopathy , Neovascularization, Pathologic/genetics , Animals , Chromogranins/genetics , Diabetic Retinopathy/genetics , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/pathology , Humans , Mice , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Retinal Vessels/metabolism , Retinal Vessels/pathology , Secretory Vesicles/genetics , Secretory Vesicles/metabolism , Secretory Vesicles/pathology
19.
J Cell Biol ; 216(9): 2945-2958, 2017 09 04.
Article in English | MEDLINE | ID: mdl-28687666

ABSTRACT

The migration of primordial germ cells (PGCs) from their place of origin to the embryonic gonad is an essential reproductive feature in many animal species. In Drosophila melanogaster, a single G protein-coupled receptor, Trapped in endoderm 1 (Tre1), mediates germ cell polarization at the onset of active migration and directs subsequent migration of PGCs through the midgut primordium. How these different aspects of cell behavior are coordinated through a single receptor is not known. We demonstrate that two highly conserved domains, the E/N/DRY and NPxxY motifs, have overlapping and unique functions in Tre1. The Tre1-NRY domain via G protein signaling is required for reading and responding to guidance and survival cues controlled by the lipid phosphate phosphatases Wunen and Wunen2. In contrast, the Tre1-NPIIY domain has a separate role in Rho1- and E-cadherin-mediated polarization at the initiation stage independent of G protein signaling. We propose that this bifurcation of the Tre1 G protein-coupled receptor signaling response via G protein-dependent and independent branches enables distinct spatiotemporal regulation of germ cell migration.


Subject(s)
Cell Polarity , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Embryonic Germ Cells/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Animals, Genetically Modified , Cadherins/genetics , Cadherins/metabolism , Cell Movement , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Genotype , Ligands , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Microscopy, Video , Mutation , Phenotype , Phosphatidate Phosphatase/genetics , Phosphatidate Phosphatase/metabolism , Protein Domains , Receptors, G-Protein-Coupled/genetics , Signal Transduction , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism
20.
Angiogenesis ; 20(4): 479-492, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28447229

ABSTRACT

Angiogenic factors play an important role in the pathogenesis of diabetic retinopathy (DR), neovascular age-related macular degeneration (nAMD) and retinopathy of prematurity (ROP). Pleiotrophin, a well-known angiogenic factor, was recently reported to be upregulated in the vitreous fluid of patients with proliferative DR (PDR). However, its pathogenic role and therapeutic potential in ocular vascular diseases have not been defined in vivo. Here using corneal pocket assays, we demonstrated that pleiotrophin induced angiogenesis in vivo. To investigate the pathological role of pleiotrophin we used neutralizing antibody to block its function in multiple in vivo models of ocular vascular diseases. In a mouse model of DR, intravitreal injection of pleiotrophin-neutralizing antibody alleviated diabetic retinal vascular leakage. In a mouse model of oxygen-induced retinopathy (OIR), which is a surrogate model of ROP and PDR, we demonstrated that intravitreal injection of anti-pleiotrophin antibody prevented OIR-induced pathological retinal neovascularization and aberrant vessel tufts. Finally, pleiotrophin-neutralizing antibody ameliorated laser-induced choroidal neovascularization, a mouse model of nAMD, suggesting that pleiotrophin is involved in choroidal vascular disease. These findings suggest that pleiotrophin plays an important role in the pathogenesis of DR with retinal vascular leakage, ROP with retinal neovascularization and nAMD with choroidal neovascularization. The results also support pleiotrophin as a promising target for anti-angiogenic therapy.


Subject(s)
Carrier Proteins/therapeutic use , Cytokines/therapeutic use , Retinal Neovascularization/drug therapy , Animals , Carrier Proteins/pharmacology , Cell Proliferation/drug effects , Choroidal Neovascularization/drug therapy , Choroidal Neovascularization/pathology , Cytokines/pharmacology , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mice, Inbred C57BL , Neovascularization, Physiologic/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Retina/drug effects , Retina/pathology , Retinal Neovascularization/pathology , Spheroids, Cellular/drug effects , Spheroids, Cellular/pathology
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