The Role of Collagen VIII in the Aging Mouse Kidney.

The gradual loss of kidney function due to increasing age is accompanied by structural changes such as fibrosis of the tissue. The underlying molecular mechanisms are complex, but not yet fully understood. Non-fibrillar collagen type VIII (COL8) could be a potential factor in the fibrosis processes of the aging kidney. A pathophysiological significance of COL8 has already been demonstrated in the context of diabetic kidney disease, with studies showing that it directly influences both the development and progression of renal fibrosis occurring. The aim of this study was to investigate whether COL8 impacts age-related micro-anatomical and functional changes in a mouse model. The kidneys of wild-type (Col8-wt) and COL8-knockout (Col8-ko) mice of different age and sex were characterized with regard to the expression of molecular fibrosis markers, the development of nephrosclerosis and renal function. The age-dependent regulation of COL8 mRNA expression in the wild-type revealed sex-dependent effects that were not observed with collagen IV (COL4). Histochemical staining and protein analysis of profibrotic cytokines TGF-ß1 (transforming growth factor) and CTGF (connective tissue growth factor) in mouse kidneys showed significant age effects as well as interactions of the factors age, sex and Col8 genotype. There were also significant age and Col8 genotype effects in the renal function data analyzed by urinary cystatin C. In summary, the present study shows, for the first time, that COL8 is regulated in an age- and sex-dependent manner in the mouse kidney and that the expression of COL8 influences the severity of age-induced renal fibrosis and function.

Exudative Age-Related Macular Degeneration: Association between Treatment Efficacy and Single-Nucleotide Variants in RAD51B, TRIB1, COL8A1, COL10A1, IL-9, IL-10, and VEGFA Genes.

Age-related macular degeneration (AMD) is a progressive neurodegenerative condition leading to vision loss and eventual blindness, with exudative AMD posing a heightened risk due to choroidal neovascularization and localized edema. Therapies targeting the VEGF pathway aim to address this mechanism for treatment effectiveness. Our study aimed to evaluate associations between specific genetic variants (RAD51B rs8017304, rs2588809; TRIB1 rs6987702, rs4351379; COL8A1 rs13095226; COL10A1 rs1064583; IL-9 rs1859430, rs2069870, rs11741137, rs2069885, rs2069884; IL-10 rs1800871, rs1800872, rs1800896; VEGFA rs1570360, rs699947, rs3025033, rs2146323) and the response to anti-VEGF treatment for exudative AMD. We enrolled 119 patients with exudative AMD categorized as responders or non-responders based on their response to anti-VEGF treatment. Statistical analysis revealed that RAD51B rs8017304 heterozygous and homozygous minor allele carriers had increased CMT before treatment compared to wild-type genotype carriers (p = 0.004). Additionally, TRIB1 rs4351379 heterozygous and homozygous minor allele carriers exhibited a greater decrease in central macular thickness (CMT) after 6 months of treatment than wild-type genotype carriers (p = 0.030). IL-9 rs1859430, rs2069870, and rs2069884 heterozygous and homozygous minor allele carriers had worse BCVA before treatment than wild-type genotype carriers (p = 0.018, p = 0.012, p = 0.041, respectively). Conversely, IL-9 rs2069885 heterozygous and homozygous minor allele carriers showed greater improvement in BCVA after 6 months compared to wild-type genotype carriers (p = 0.032). Furthermore, VEGFA rs699947 heterozygous and homozygous minor allele carriers had better BCVA before treatment and after 3 and 6 months of treatment than wild-type genotype carriers (p = 0.003, p = 0.022, respectively), with these carriers also exhibiting higher CMT after 6 months of anti-VEGF treatment (p = 0.032). Not all results remained statistically significant under this stringent correction for multiple comparisons. The comparisons of the serum concentrations of IL-10, VEGF-A, and VEGF-R2/KDR between non-responders and responders did not yield statistically significant differences. Our study identified significant associations between genetic variants, including RAD51B rs8017304, TRIB1 rs4351379, IL-9 rs1859430, rs2069870, rs2069884, rs2069885, and VEGFA rs699947, and parameters related to the efficacy of exudative AMD treatment, such as BCVA and CMT.

COL8A1 facilitates the growth of triple-negative breast cancer via FAK/Src activation.

PURPOSE: Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes, and treatment options are limited because of the lack of signature molecules and heterogeneous properties of cancer. COL8A1 expression is higher in breast cancer than in normal tissues and is strongly correlated with worse overall survival in patients with breast cancer. However, the biological function of COL8A1 on cancer progression is not fully understood. In this study, we investigated the biological function of COL8A1 on TNBC progression. METHODS: COL8A1-deficient cells were generated using the CRISPR-Cas9 system. The tumor growth and metastasis of TNBC cells were evaluated using three-dimensional culture (3D) methods and xenograft mouse models. The activation of focal adhesion kinase (FAK)/Src by COL8A1 in TNBC cells was evaluated by immunoblotting. RESULTS: COL8A1 expression was primarily distributed into TNBC cell lines. Further, relapse-free survival in TNBC patients with the MSL subtype was strongly associated with the COL8A1 expression. MDA-MB-231 and Hs578T cells, classified as the MSL subtype, strongly express COL8A1, and COL8A1 protein expression was induced by hypoxia in both cell lines. Loss of COL8A1 expression inhibited spheroid /tumor growth and metastasis in vitro and in vivo. Further, exogenous COL8A1 promoted TNBC growth via the FAK/Src activation. Finally, the spheroid growth of MDA-MB-231 and Hs578T cells was inhibited by defactinib, a FAK inhibitor, without cytotoxicity. CONCLUSION: These results indicate that COL8A1-mediated FAK/Src activation produces a more aggressive phenotype in TNBC, and its target inhibition may be an efficacious treatment for TNBC.

Collagen type VIII alpha 2 chain (COL8A2), an important component of the basement membrane of the corneal endothelium, facilitates the malignant development of glioblastoma cells via inducing EMT.

Glioblastoma (GBM) is one of the most lethal tumor of all human cancers. Due to its poor response to chemotherapy and radiotherapy as well as its high rate of recurrence after treatment, the treatment is still undesired. The identification of potential related genes and bio-markers in the development of GBM could provide some new targets for the treatment of GBM. Our purpose in this study was to evaluate the mission of COL8A2 in GBM. Combined with TCGA, Oncomine databases, CGGA, GEPIA website and qRT-PCR analyses, we found that COL8A2 was up-regulated both in GBM tissues and cells compared to the controls. Moreover, the high COL8A2 expression was associated with the shorter overall survival of patients with GBM. The expression of COL8A2 was also positively correlated with metastasis-associated genes including vimentin, snail, slug, MMP2 and MMP7 according to GEPIA website. Knockdown of COL8A2 could suppress the cell proliferation, cell migration and invasion, whereas the overexpression of COL8A2 significantly expedited these processes. What's more, the outcome of western blot analysis manifested that COL8A2 could induced the expression of vimentin, snail, slug, MMP2 and MMP7. Taken together, COL8A2 activated cell proliferation, cell migration and invasion via raising the relative expression of EMT-related proteins in GBM. Therefore, our investigation suggests the oncogenic role of COL8A2 in GBM and provides a potential application of COL8A2 for GBM therapy.

Functional Impact of Human Genetic Variants of COL18A1/Endostatin on Pulmonary Endothelium.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by disordered and dysfunctional angiogenesis leading to small-vessel loss and an obliterative vasculopathy. The pathogenesis of PAH is not fully understood, but multiple studies have demonstrated links between elevated angiostatic factors, disease severity, and adverse clinical outcomes. ES (endostatin), one such circulating angiostatic peptide, is the cleavage product of the proteoglycan COL18A1 (collagen α1[XVIII] chain). Elevated serum ES is associated with increased mortality and disease severity in PAH. A nonsynonymous variant of ES (aspartic acid-to-asparagine substitution at amino acid 104; p.D104N) is associated with differences in PAH survival. Although COL18A1/ES expression is markedly increased in remodeled pulmonary vessels in PAH, the impact of ES on pulmonary endothelial cell (PEC) biology and molecular contributions to PAH severity remain undetermined. In the present study, we characterized the effects of exogenous ES on human PEC biology and signaling. We demonstrated that ES inhibits PEC migration, proliferation, and cell survival, with significant differences between human variants, indicating that they are functional genetic variants. ES promotes proteasome-mediated degradation of the transcriptional repressor ID1, increasing expression and release of TSP-1 (thrombospondin 1). ES inhibits PEC migration via an ID1/TSP-1/CD36-dependent pathway, in contrast to proliferation and apoptosis, which require both CD36 and CD47. Collectively, the data implicate ES as a novel negative regulator of ID1 and an upstream propagator of an angiostatic signal cascade converging on CD36 and CD47, providing insight into the cellular and molecular effects of a functional genetic variant linked to altered outcomes in PAH.

COL18A1 is a candidate eye iridocorneal angle-closure gene in humans.

Primary angle-closure glaucoma (PACG) is a common form of glaucoma in the Far East. Its defining feature is iridocorneal angle closure. In addition to PACG, indications of angle closure are included in the diagnostic criteria of related conditions primary angle-closure suspect (PACS) and primary angle closure (PAC). To the best of our knowledge, a causative gene for iridocorneal angle closure in humans has not been identified. This study aimed to identify the genetic cause of iridocorneal angle closure in a pedigree with at least 10 individuals diagnosed with PACS, PAC or PACG. Results of linkage analysis, segregation analysis of 44 novel variations, whole exome sequencing of 10 individuals, screenings of controls and bioinformatics predictions identified a mutation in COL18A1 that encodes collagen type XVIII as the most likely cause of angle closure in the pedigree. The role of COL18A1 in the etiology of Knobloch syndrome (KS) that is consistently accompanied by optic anomalies, available functional data on the encoded protein and the recognized role of collagens and the extracellular matrix in glaucoma pathogenesis supported the proposed role of the COL18A1 mutation in the pedigree. Subsequent identification of other COL18A1 mutations in PACS affected individuals of two unrelated families further supported that COL18A1 may affect angle closure. These PACS individuals were parents and grandparents of KS-affected children. In conclusion, a gene that affects angle closure in humans, a critical feature of PACG, has been identified. The findings also reinforce the importance of collagens in eye features and functions.

Zonal regulation of collagen-type proteins and posttranslational modifications in prostatic benign and cancer tissues by imaging mass spectrometry.

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.

RNAseq-Based Prioritization Revealed COL6A5, COL8A1, COL10A1 and MIR146A as Common and Differential Susceptibility Biomarkers for Psoriasis and Psoriatic Arthritis: Confirmation from Genotyping Analysis of 1417 Italian Subjects.

Psoriasis (Ps) and Psoriatic Arthritis (PsA) are characterized by a multifactorial etiology, involving genetic and environmental factors. The present study aimed to investigate polymorphisms (SNPs) within genes involved in extracellular matrix and cell homeostasis and microRNA genes as susceptibility biomarkers for Ps and PsA. Bioinformatic analysis on public RNA-seq data allowed for selection of rs12488457 (A/C, COL6A5), rs13081855 (G/T, COL8A1), rs3812111 (A/T, COL10A1) and rs2910164 (C/G, MIR146A) as candidate biomarkers. These polymorphisms were analyzed by Real-Time PCR in a cohort of 1417 Italian patients (393 Ps, 424 PsA, 600 controls). Statistical and bioinformatic tools were utilized for assessing the genetic association and predicting the effects of the selected SNPs. rs12488457, rs13081855 and rs2910164 were significantly associated with both Ps (p = 1.39 × 10-8, p = 4.52 × 10-4, p = 0.04, respectively) and PsA (p = 5.12 × 10-5, p = 1.19 × 10-6, p = 0.01, respectively). rs3812111, instead, was associated only with PsA (p = 0.005). Bioinformatic analysis revealed common and differential biological pathways involved in Ps and PsA. COL6A5 and COL8A1 take part in the proliferation and angiogenic pathways which are altered in Ps/PsA and contribute to inflammation together with MIR146A. On the other hand, the exclusive association of COL10A1 with PsA highlighted the specific involvement of bone metabolism in PsA.

Biomechanical changes to Descemet's membrane precede endothelial cell loss in an early-onset murine model of Fuchs endothelial corneal dystrophy.

Early-onset Fuchs endothelial corneal dystrophy (FECD) has been associated with nonsynonymous mutations in collagen VIII α2 (COL8A2), a key extracellular matrix (ECM) protein in Descemet's membrane (DM). Two knock-in strains of mice have been generated to each express a mutant COL8A2 protein (Col8a2L450W/L450W and Col8a2Q455K/Q455K) that recapitulate the clinical phenotype of early-onset FECD including endothelial cell loss, cellular polymegathism and pleomorphism, and guttae. Due to abnormalities in ECM protein composition and structure in FECD, the stiffness of DM in Col8a2 knock-in mice and wildtype (WT) controls was measured using atomic force microscopy at 5 and 10 months of age, coinciding with the onset of FECD phenotypic abnormalities. At 5 months, only sporadic guttae were identified via in vivo confocal microscopy (IVCM) in Col8a2Q455K/Q455K mice, otherwise both strains of Col8a2 transgenic mice were indistinguishable from WT controls in terms of endothelial cell density and size. By 10 months of age, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice developed reduced corneal endothelial density, increased endothelial cell area and guttae, with the Col8a2Q455K/Q455K strain exhibiting a more severe phenotype. However, at 5 months of age, prior to the development endothelial cell abnormalities, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice knock-in mice had reduced tissue stiffness of DM that was statistically significant in the Col8a2Q455K/Q455K mice when compared with wildtype controls. These data indicate that alterations in the tissue compliance of DM precede phenotypic changes in endothelial cell count and morphology, and may play a role in onset and progression of FECD.

Type XVIII Collagen Modulates Keratohyalin Granule Formation and Keratinization in Oral Mucosa.

Epithelial keratinization involves complex cellular modifications that provide protection against pathogens and chemical and mechanical injuries. In the oral cavity, keratinized mucosa is also crucial to maintain healthy periodontal or peri-implant tissues. In this study, we investigated the roles of type XVIII collagen, a collagen-glycosaminoglycan featuring an extracellular matrix component present in the basement membrane, in oral mucosal keratinization. Histological analysis of keratinized and non-keratinized oral mucosa showed that type XVIII collagen was highly expressed in keratinized mucosa. Additionally, a 3D culture system using human squamous carcinoma cells (TR146) was used to evaluate and correlate the changes in the expression of type XVIII collagen gene, COL18A1, and epithelial keratinization-related markers, e.g., keratin 1 (KRT1) and 10 (KRT10). The results showed that the increase in COL18A1 expression followed the increase in KRT1 and KRT10 mRNA levels. Additionally, loss-of-function analyses using silencing RNA targeting COL18A1 mRNA and a Col18-knockout (KO) mouse revealed that the absence of type XVIII collagen induces a dramatic decrease in KRT10 expression as well as in the number and size of keratohyalin granules. Together, the results of this study demonstrate the importance of type XVIII collagen in oral mucosal keratinization.

Comparative gene array analyses of severe elastic fiber defects in late embryonic and newborn mouse aorta.

Elastic fibers provide reversible elasticity to the large arteries and are assembled during development when hemodynamic forces are increasing. Mutations in elastic fiber genes are associated with cardiovascular disease. Mice lacking expression of the elastic fiber genes elastin ( Eln-/-), fibulin-4 ( Efemp2-/-), or lysyl oxidase ( Lox-/-) die at birth with severe cardiovascular malformations. All three genetic knockout models have elastic fiber defects, aortic wall thickening, and arterial tortuosity. However, Eln-/- mice develop arterial stenoses, while Efemp2-/- and Lox-/- mice develop ascending aortic aneurysms. We performed comparative gene array analyses of these three genetic models for two vascular locations and developmental stages to determine differentially expressed genes and pathways that may explain the common and divergent phenotypes. We first examined arterial morphology and wall structure in newborn mice to confirm that the lack of elastin, fibulin-4, or lysyl oxidase expression provided the expected phenotypes. We then compared gene expression levels for each genetic model by three-way ANOVA for genotype, vascular location, and developmental stage. We found three genes upregulated by genotype in all three models, Col8a1, Igfbp2, and Thbs1, indicative of a common response to severe elastic fiber defects in developing mouse aorta. Genes that are differentially regulated by vascular location or developmental stage in all three models suggest mechanisms for location or stage-specific disease pathology. Comparison of signaling pathways enriched in all three models shows upregulation of integrins and matrix proteins involved in early wound healing, but not of mature matrix molecules such as elastic fiber proteins or fibrillar collagens.

Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.

BACKGROUND: Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis. METHODS: We analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with ß1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student's t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study. RESULTS: Collagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced ß1 integrin activation. Blocking ß1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis. CONCLUSIONS: Our results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.

Activation of JNK signaling in osteoblasts is inversely correlated with collagen synthesis in age-related osteoporosis.

The age-related reduction in the function of osteoblasts plays a central role in the pathogenesis of bone loss and osteoporosis. Collagen synthesis is a primary function of differentiated osteoblasts, however, the mechanisms for age-related changes in collagen synthesis in human osteoblasts remain elusive. We use Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) analysis to exploit the transcriptional profiles of osteoblasts from young and old donors. A panel of collagen members was downregulated in aged osteoblasts, including COL12A1, COL5A1, COL5A3, COL8A1 and COL8A2. Co-expression analysis followed by GO analysis revealed that oxidoreductase activity and kinase activity were inversely correlated with collagen synthesis in osteoblasts. GESA analysis further showed that JNK signaling was upregulated in aged osteoblasts. Consistently, MAP3K4 and MAP4K2, upstream of JNK, were also increased in aged osteoblasts. Moreover, expression levels of MAP3K4 were significantly inversely correlated with levels of the collagen genes. Those transcriptomic results were further verified by examining clinical specimens of osteoporosis by immunohistochemistry. These results provide transcriptomic evidence that deregulated JNK signaling may impair collagen synthesis in osteoblasts and imply a therapeutic value of JNK inhibitors for treating osteoporosis and preventing skeletal aging by counteracting the age-related reduction in the function of osteoblasts.

Whole-Exome Sequencing in Age-Related Macular Degeneration Identifies Rare Variants in COL8A1, a Component of Bruch's Membrane.

PURPOSE: Genome-wide association studies and targeted sequencing studies of candidate genes have identified common and rare variants that are associated with age-related macular degeneration (AMD). Whole-exome sequencing (WES) studies allow a more comprehensive analysis of rare coding variants across all genes of the genome and will contribute to a better understanding of the underlying disease mechanisms. To date, the number of WES studies in AMD case-control cohorts remains scarce and sample sizes are limited. To scrutinize the role of rare protein-altering variants in AMD cause, we performed the largest WES study in AMD to date in a large European cohort consisting of 1125 AMD patients and 1361 control participants. DESIGN: Genome-wide case-control association study of WES data. PARTICIPANTS: One thousand one hundred twenty-five AMD patients and 1361 control participants. METHODS: A single variant association test of WES data was performed to detect variants that are associated individually with AMD. The cumulative effect of multiple rare variants with 1 gene was analyzed using a gene-based CMC burden test. Immunohistochemistry was performed to determine the localization of the Col8a1 protein in mouse eyes. MAIN OUTCOME MEASURES: Genetic variants associated with AMD. RESULTS: We detected significantly more rare protein-altering variants in the COL8A1 gene in patients (22/2250 alleles [1.0%]) than in control participants (11/2722 alleles [0.4%]; P = 7.07×10-5). The association of rare variants in the COL8A1 gene is independent of the common intergenic variant (rs140647181) near the COL8A1 gene previously associated with AMD. We demonstrated that the Col8a1 protein localizes at Bruch's membrane. CONCLUSIONS: This study supported a role for protein-altering variants in the COL8A1 gene in AMD pathogenesis. We demonstrated the presence of Col8a1 in Bruch's membrane, further supporting the role of COL8A1 variants in AMD pathogenesis. Protein-altering variants in COL8A1 may alter the integrity of Bruch's membrane, contributing to the accumulation of drusen and the development of AMD.

Effects of COL8A1 on the proliferation of muscle-derived satellite cells.

Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P-AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3 K/AKT signaling pathway.

Co-expression Network Analysis Identified COL8A1 Is Associated with the Progression and Prognosis in Human Colon Adenocarcinoma.

BACKGROUNDS AND AIMS: Human colon adenocarcinoma is one of the major causes of tumor-induced death worldwide. A complicated gene interconnection network significantly regulates its progression and prognosis. The aim of our study was to find hub genes associated with the progression and prognosis of colon adenocarcinoma and to illustrate the underlying mechanisms. METHODS: A weighted gene co-expression network analysis was performed in our study to identify significant gene modules and hub genes associated with the TNM stage of colon adenocarcinoma (n = 441). RESULTS: In the turquoise module of interest, 23 hub genes were initially selected, and 10 of them were identified as "real" hub genes with high connectivity in the protein-protein interaction network. In the terms of validation, COL8A1 had the highest correlation with clinical traits among all of the hub genes. Data obtained from the Oncomine and GEPIA databases showed a higher expression of COL8A1 in colon adenocarcinoma tissues compared with normal colon tissues. Kaplan-Meier survival curves showed that higher expression of COL8A1 resulted in a shorter overall survival time and disease-free survival time. Univariate and multivariate Cox proportional hazards analyses indicated that the COL8A1 expression was an independent prognostic factor for survival in colon adenocarcinoma patients. Finally, gene set enrichment analysis indicated that the gene sets associated with focal adhesion were significantly enriched in colon adenocarcinoma samples with COL8A1 highly expressed. CONCLUSIONS: COL8A1 was identified and proved to be correlated with the progression and prognosis of human colon adenocarcinoma, probably through regulating focal adhesion-related pathways.

Enhanced expression of Vastatin inhibits angiogenesis and prolongs survival in murine orthotopic glioblastoma model.

BACKGROUND: Antiangiogenic therapies are considered promising for the treatment of glioblastoma (GB). The non-collagenous C-terminal globular NC1 domain of type VIII collagen a1 chain, Vastatin, is an endogenous antiangiogenic polypeptide. Sustained enhanced expression of Vastatin was shown to inhibit tumour growth and metastasis in murine hepatocellular carcinoma models. In this study, we further explored the efficacy of Vastatin in the treatment of GB xenografts. METHOD: Treatment of Vastatin was carried out using a nanopolymer gene vector PEI600-CyD-Folate (H1). Antiangiogenic effect of Vastatin was tested in vitro by using co-culture system and conditioned medium. An orthotopic GB murine model was established to examine the in vivo therapeutic effect of Vastatin alone treatment and its combination with temozolomide. RESULTS: Vastatin gene transfection mediated by H1 could target tumour cells specifically and suppress the proliferation of microvessel endothelial cells (MECs) through a paracrine inhibition manner. Enhancing Vastatin expression by intracerebral injection of H1-Vastatin significantly prolonged animal survival from 48 to 75 days in GB murine model, which was comparable to the effect of Endostatin, the most studied endogenous antiangiogenic polypeptide. The diminished presence of CD34 positive cells in the GB xenografts suggested that Vastatin induced significant antiangiogenesis. Moreover, a synergistic effect in extending survival was detected when H1-Vastatin was administered with temozolomide (TMZ) in GB chemoresistant murine models. CONCLUSION: Our results suggest, for the first time, that Vastatin is an antiangiogenic polypeptide with significant potential therapeutic benefit for GB. H1-Vastatin gene therapy may have important implications in re-sensitizing recurrent GB to standard chemotherapeutic agents.

Vastatin, an Endogenous Antiangiogenesis Polypeptide That Is Lost in Hepatocellular Carcinoma, Effectively Inhibits Tumor Metastasis.

Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.

2,3,7,8 Tetrachlorodibenzo-p-dioxin-induced RNA abundance changes identify Ackr3, Col18a1, Cyb5a and Glud1 as candidate mediators of toxicity.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is an aromatic, long-lived environmental contaminant. While the pathogenesis of TCDD-induced toxicity is poorly understood, it has been shown that the aryl hydrocarbon receptor (AHR) is required. However, the specific transcriptomic changes that lead to toxic outcomes have not yet been identified. We previously identified a panel of 33 genes that respond to TCDD treatment in two TCDD-sensitive rodent species. To identify genes involved in the onset of hepatic toxicity, we explored 25 of these in-depth using liver from two rat strains: the TCDD-resistant Han/Wistar (H/W) and the TCDD-sensitive Long-Evans (L-E). Time course and dose-response analyses of mRNA abundance following TCDD insult indicate that eight genes are similarly regulated in livers of both strains of rat, suggesting that they are not central to the severe L-E-specific TCDD-induced toxicities. The remaining 17 genes exhibited various divergent mRNA abundances between L-E and H/W strains after TCDD treatment. Several genes displayed a biphasic response where the initial response to TCDD treatment was followed by a secondary response, usually of larger magnitude in L-E liver. This secondary response was most often an exaggeration of the original TCDD-induced response. Only cytochrome b5 type A (microsomal) (Cyb5a) had equivalent TCDD sensitivity to the prototypic AHR-responsive cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1), while six genes were less sensitive. Four genes showed an early inter-strain difference that was sustained throughout most of the time course (atypical chemokine receptor 3 (Ackr3), collagen, type XVIII, alpha 1 (Col18a1), Cyb5a and glutamate dehydrogenase 1 (Glud1)), and of those genes examined in this study, are most likely to represent genes involved in the pathogenesis of TCDD-induced hepatotoxicity in L-E rats.

Mutational spectrum of Korean patients with corneal dystrophy.

Corneal dystrophy typically refers to a group of rare hereditary disorders with a heterogeneous genetic background. A comprehensive molecular genetic analysis was performed to characterize the genetic spectrum of corneal dystrophies in Korean patients. Patients with various corneal dystrophies underwent thorough ophthalmic examination, histopathologic examination, and Sanger sequencing. A total of 120 probands were included, with a mean age of 50 years (SD = 18 years) and 70% were female. A total of 26 mutations in five genes (14 clearly pathogenic and 12 likely pathogenic) were identified in 49 probands (41%). Epithelial-stromal TGFBI dystrophies, macular corneal dystrophy and Schnyder corneal dystrophy (SCD) showed 100% mutation detection rates, while endothelial corneal dystrophies showed lower detection rates of 3%. Twenty six non-duplicate mutations including eight novel mutations were identified and mutations associated with SCD were identified genetically for the first time in this population. This study provides a comprehensive characterization of the genetic aberrations in Korean patients and also highlights the diagnostic value of molecular genetic analysis in corneal dystrophies.