Authentication of a novel antibody to zebrafish collagen type XI alpha 1 chain (Col11a1a)

Objective Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). Results The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.

Proteomic Analysis Reveals Key Proteins in Extracellular Vesicles Cargo Associated with Idiopathic Pulmonary Fibrosis In Vitro

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and highly fatal disease. It is characterized by the increased activation of both fibroblast and myofibroblast that results in excessive extracellular matrix (ECM) deposition. Extracellular vesicles (EVs) have been described as key mediators of intercellular communication in various pathologies. However, the role of EVs in the development of IPF remains poorly understood. This study aimed to characterize the differentially expressed proteins contained within EVs cargo derived from the fibroblast cell lines LL97A (IPF-1) and LL29 (IPF-2) isolated from lungs bearing IPF as compared to those derived from the fibroblast cell lines CCD8Lu (NL-1) and CCD19Lu (NL-2) isolated from healthy donors. Isolated EVs were subjected to label-free quantitative proteomic analysis by LC-MS/MS, and as a result, 331 proteins were identified. Differentially expressed proteins were obtained after the pairwise comparison, including all experimental groups. A total of 86 differentially expressed proteins were identified in either one or more comparison groups. Of note, proteins involved in fibrogenic processes, such as tenascin-c (TNC), insulin-like-growth-factor-binding protein 7 (IGFBP7), fibrillin-1 (FBN1), alpha-2 collagen chain (I) (COL1A2), alpha-1 collagen chain (I) (COL1A1), and lysyl oxidase homolog 1 (LOXL1), were identified in EVs cargo isolated from IPF cell lines. Additionally, KEGG pathway enrichment analysis revealed that differentially expressed proteins participate in focal adhesion, PI3K-Akt, and ECM–receptor interaction signaling pathways. In conclusion, our findings reveal that proteins contained within EVs cargo might play key roles during IPF pathogenesis.

Somatostatin Therapy Improves Stellate Cell Activation and Early Fibrogenesis in a Preclinical Model of Extended Major Hepatectomy

Liver resection treats primary and secondary liver tumors, though clinical applicability is limited by the remnant liver mass and quality. Herein, major hepatic resections were performed in pigs to define changes associated with sufficient and insufficient remnants and improve liver-specific outcomes with somatostatin therapy. Three experimental groups were performed: 75% hepatectomy (75H), 90% hepatectomy (90H), and 90% hepatectomy + somatostatin (90H + SST). Animals were followed for 24 h (N = 6) and 5 d (N = 6). After hepatectomy, portal pressure gradient was higher in 90H versus 75H and 90H + SST (8 (3–13) mmHg vs. 4 (2–6) mmHg and 4 (2–6) mmHg, respectively, p < 0.001). After 24 h, changes were observed in 90H associated with stellate cell activation and collapse of sinusoidal lumen. Collagen chain type 1 alpha 1 mRNA expression was higher, extracellular matrix width less, and percentage of collagen-staining areas greater at 24 h in 90H versus 75H and 90H + SST. After 5 d, remnant liver mass was higher in 75H and 90H + SST versus 90H, and Ki-67 immunostaining was higher in 90H + SST versus 75H and 90H. As well, more TUNEL-staining cells were observed in 90H versus 75H and 90H + SST at 5 d. Perioperative somatostatin modified portal pressure, injury, apoptosis, and stellate cell activation, stemming changes related to hepatic fibrogenesis seen in liver remnants not receiving treatment.

Authentication of a novel antibody to zebrafish collagen type XI alpha 1 chain (Col11a1a)

Objective Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). Results The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Coll11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.

The extracellular matrix complexity of idiopathic epiretinal membranes and the bilaminar arrangement of the associated internal limiting membrane in the posterior retina

Purpose To study the composition of the internal limiting membrane (ILM) of the retina, the extracellular matrix (ECM) of idiopathic epiretinal membranes (iERMs), and the relationships occurring between the two membranes. Methods Forty-six iERMs, 24 of them associated with the ILM, were collected and included in this study. The investigation has been carried out by immunofluorescence and confocal microscopy on glutaraldehyde- and osmium-fixed epon-embedded samples and on frozen samples. Sections were double or triple labelled with antibodies against vimentin; collagens I, III, IV, α5(IV), and VI; laminin 1 + 2; laminin α2-, α4-, α5-, β1-, β2-, β3-, γ1-, and γ2-chains; entactin; and fibronectin. Results iERM thickness was not uniform. Almost 14% of iERMs showed thickenings due to folding of their ECM component under the cell layer. The vitreal side of iERMs was often shorter than the attached ILM. In this case, the ILM resulted folded under the iERM. ILMs contained laminin 111; laminin α2-, α5-, β1-, β2-, and γ1-chains; entactin; collagens I; α5(IV); [α1(IV)]2α2(IV); and VI. Laminins, entactin, and α5(IV) were gathered on the retinal half of the ILM, whereas collagens [α1(IV)]2α2(IV) and I were restricted to the vitreal side. Collagen VI was detected on both sides of the ILM. iERMs expressed laminin 111, collagens III, [α1(IV)]2α2(IV) and VI, entactin, and fibronectin. Entactin co-localized with laminins and collagen IV. Conclusions Analysis of laminins and collagen chain expression indicates that ILM contains laminin 111 (former laminin 1), laminin 521 (former laminin 11), laminin 211 (former laminin 2), collagen [α1(IV)]2α2(IV), and collagen α3(IV)α4(IV)α5. In contrast, iERMs express only collagen [α1(IV)]2α2(IV) and laminin 111. In addition, both iERMs and ILMs contain entactin. The presence of three major constituents of the basement membranes co-localized together in iERMs is suggestive for a deranged process of basement membrane formation which fails to assemble properly. In view of the many interactions occurring among its proteins, the ECM of either the iERMs or the ILMs can account for their reciprocal adhesiveness. In addition, the peculiar deposition of the ECM observed in some samples of iERM is suggestive for its involvement in the formation of macular puckers.

Вплив поліморфізму генів PTHR1 та COL1A1 на розвиток захворювань тканин пародонта у людей молодого віку

At the present stage of development of dentistry, the leading Ukrainian and foreign scientists devote a considerable part of the research to a deeper study of the etiology and pathogenetic mechanisms of periodontal tissue diseases and the influence of various exogenous and endogenous factors on their course.The aim of the study – to learn the ability and methods of forecasting and early diagnosis of the periodontal tissue lesions in young people. Materials and Methods. During our research we examined 24 young people with periodontal tissue diseases, who were included to the main group, and 15 healthy people, who formed the control group. The complex clinical examination was performed in each research group. It was studied the distribution of polymorphous variants of the type I parathormone receptor and the α1-chain of collagen gene with a help of polymerase chain reaction by restrictase cleavage of DNA fragments and electrophoresis in polyacrylamide gel (AA/BA 29:1). Results and Discussion. The distribution of genotypes by PTHR1 gene in control group was similar to those in main group (p>0.05). Also we didn’t find the difference between frequencies of the separate alleles in people with periodontal tissue pathology and without it (p>0.05). Yes, the repetitions of the allele 5 encoding normal type I parathormone receptor were found more often, comparing with the allele 6 that is responsible for the formation of unfunctional PTHR1 (р<0.001) in both main and control groups. The dominance of the genotype TT, which corresponds to the pathology, was found in young people with the periodontal tissue lesions – (38.46 ± 4.79) %, while among the control group the genotype of norm GG was met the most often – (68.24±5.08) %. Also, the frequency of repetitions of the allele T encoding the imperfect collagen chain was (57.60±3.79) % in young people with periodontal diseases, and in the control group this figure was (13.27±2.81) %, p<0.001. Conclusions. According to our results the presence of allele T and genotype TT that correspond the imperfect collagen chain may be one of the causes of periodontal tissue pathology.

The musculoskeletal effect of exercise and soluble activin receptor type 2b in mouse models of osteogenesis imperfecta

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Osteogenesis Imperfecta (OI) is a heritable connective tissue disorder primarily due to mutations in the type I collagen containing tissues, such as bone, skin and blood vessels. The clinical manifestations of OI include skeletal deformity and fragility, scoliosis, growth retardation, and hearing loss. There is no cure for OI and current treatment is limited with anti-resorptive drugs, the bisphosphonates, and/or surgical intervention, which comes with adverse side-effects and high risk of device failure, respectively. Thus identifying an alternative strategy to strengthen the skeletal properties of OI is still critically needed. Bone and muscle are intimate tissues in a relation to their proximate locations and biochemical cross-talks. Bone responds and adapts to external stimuli, mainly the muscle mass and contractile strength, to alter its shape and mass due to its mechanosensing characteristic, and this makes muscle and bone mass to be positively correlated in normal condition. As a potential therapeutic option, we sought to enhance the muscle mass and function via physical exercise to indirectly improve the bone properties in OI. We have investigated the effects of the threadmill exercise in G610C OI mouse model, which has a glycine to cysteine substitution at position 610 of pro[alpha]2 collagen chain and mimics the phenotype of mild type I/IV OI individuals. Treadmill exercised heterozygous G610C (+/G610C) mice exhibited similar exercise capacity as wildtype littermates and had increased femoral stiffness without altering bone biomechanical strength. Muscle mass can be regulated by myostatin, a negative regulator of muscle growth, and deficiency of myostatin in mice lead to abnormal muscle fiber growth. As an alternative approach, we have investigated the effects of pharmacological myostatin inhibition by using a soluble fusion protein activin receptor type IIB-mFc (sActRIIB-mFc). Myostatin signals through activin receptor type IIB (ActRIIB) on cell surface to regulate downstream signaling pathways and the sActRIIB-mFc act as "ligand trap" to bind any circulating myostatin proteins and prevent them from binding to their endogenous cellular receptors. As first part of this study, we investigated the effects of sActRIIB-mFc on muscle properties of two molecularly distinct OI mouse models, G610C and oim. Unlike G610C mouse model, homozygous oim (oim/oim) has a mutation in col1[alpha]2 genes thus synthesizing nonfunctional pro[alpha]2(I) collagen chain and leading to synthesis of homotrimeric [alpha]1(I)3, instead of normal heterotrimeric [alpha]1(I)2[alpha]2(I). oim/oim also exhibit muscle atrophy with compromised muscle contractile strength. 8 weeks of bi-weekly sActRIIB-mFc (10mg/kg) treatment in +/G610C and oim/oim mice was able to induce the increase in body weight and skeletal muscle mass. In addition, oim/oim mice exhibited increase in absolute contractile strength without altering relative and specific muscle function, suggesting a potential therapeutic option for muscle weakness in oim/oim mice. As second part of this study, we investigated the effects of sActRIIB-mFc on skeletal properties of these two OI mouse models. sActRIIB-mFc treated +/G610C and oim/oim mice exhibited increase in trabecular bone microarchitecture, and +/G610C mice had further increase in cortical bone geometry and biomechanical strength. Overall, my current study demonstrated that sActRIIB-mFc treatment was effective in both G610C and oim mouse models to enhance their muscle and bone properties, although they exhibited different responses in such that G610C mice did not show a statistically significant increase in muscle contractile function while the oim mice did not show increase in cortical bone geometry and biomechanical strength. I postulate that this was potentially due to the differences in molecular mutation and severity of the phenotype, thus more thorough investigation in molecular and cellular mechanisms of sActRIIB-mFc in these two different OI mouse models will hold promise in developing more targeted therapeutic option for OI.