A Mouse Model of Brittle Cornea Syndrome caused by mutation in Zfp469

Brittle Cornea Syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene, Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the type I:type V collagen ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of type I collagen offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus where the cornea thins progressively.

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Collagen fibrillogenesis in vitro: interaction of types I and V collagen

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142670 ◽

1986 ◽

Vol 44 ◽

pp. 210-211

Author(s):

Arthur J. Wasserman ◽

Kathy C. Kloos ◽

David E. Birk

Keyword(s):

Type I Collagen ◽

Corneal Stroma ◽

Minor Component ◽

Homogeneous Distribution ◽

Type I ◽

Type V Collagen ◽

Fibril Morphology ◽

Type V ◽

In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

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Relative rates of biosynthesis of collagen type I, type V and type VI in calf cornea

Biochemical Journal ◽

10.1042/bj2740615 ◽

1991 ◽

Vol 274 (2) ◽

pp. 615-617 ◽

Cited By ~ 32

Author(s):

P Kern ◽

M Menasche ◽

L Robert

Keyword(s):

Type I Collagen ◽

Collagen Type ◽

Corneal Stroma ◽

Collagen Type I ◽

Type I ◽

Type Vi Collagen ◽

Sds Page ◽

Proline Incorporation ◽

Type V

The biosynthesis of type I, type V and type VI collagens was studied by incubation of calf corneas in vitro with [3H]proline as a marker. Pepsin-solubilized collagen types were isolated by salt fractionation and quantified by SDS/PAGE. Expressed as proportions of the total hydroxyproline solubilized, corneal stroma comprised 75% type I, 8% type V and 17% type VI collagen. The rates of [3H]proline incorporation, linear up to 24 h for each collagen type, were highest for type VI collagen and lowest for type I collagen. From pulse-chase experiments, the calculated apparent half-lives for types I, V and VI collagens were 36 h, 10 h and 6 h respectively.

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Evaluation In Vivo and In Vitro of the Antioxidant, Antinociceptive, and Anti-Inflammatory Activities of Biflavonoids From Ouratea hexasperma and O. ferruginea

Natural Product Communications ◽

10.1177/1934578x19856802 ◽

2019 ◽

Vol 14 (6) ◽

pp. 1934578X1985680 ◽

Cited By ~ 1

Author(s):

Poliana de Araujo Oliveira ◽

Queli Cristina Fidelis ◽

Thayane Ferreira da Costa Fernandes ◽

Milene Conceição de Souza ◽

Dayane Magalhães Coutinho ◽

...

Keyword(s):

Type I Collagen ◽

In Vivo Model ◽

Type I ◽

Anti Inflammatory ◽

Vitro Model ◽

Factor Α ◽

Necrosis Factor

Ouratea species are used for the treatment of inflammation-related diseases such as rheumatism and arthritic disorders. The Ouratea genus is a rich source of flavonoids and bioflavonoids and for this reason we evaluated the effects of the biflavonoid fractions from the leaves of O. hexasperma (OHME) and O. ferruginea (OFME) in the in vivo model of complete Freund’s adjuvant (CFA)-induced arthritis and in the in vitro model of oxidative stress and cellular viability. The CFA-induced arthritis model in rats was followed by paw volume, articular incapacitation and Randall-selitto models, as well as quantification of cytokines and serum C-terminal telopeptide of type I collagen levels. OHME and OFME demonstrated antinociceptive and anti-inflammatory activities, as well as improvement in articular incapacity and reduction in levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α, and type 1 collagen, and increased cell viability. No adverse effects were observed. The results suggest that OHME and OFME can reduce inflammation and bone resorption besides their antioxidant action.

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Surface Modified Poly(vinyl alcohol) Nanofiber for the Artificial Corneal Stroma

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.342-343.209 ◽

2007 ◽

Vol 342-343 ◽

pp. 209-212 ◽

Cited By ~ 4

Author(s):

Hisatoshi Kobayashi

Keyword(s):

Corneal Epithelium ◽

Vinyl Alcohol ◽

Type I Collagen ◽

Corneal Stroma ◽

Light Transmittance ◽

Poly Vinyl Alcohol ◽

Type I ◽

Nanofiber Sheets

Previously we have found that the immobilization of Type I collagen on the poly(vinyl alcohol)(PVA) hydrogel disc was effective in supporting adhesion and growth of the corneal epithelium and stromal cell in vitro. But the durability of the produced corneal epithelium layer in vivo has some problem. We hypothesized the cell construction force is much stronger than the force of the cell adhesion on the flat modified PVA surfaces. Therefore the improvement of mechanical anchoring force between the substrate and formed corneal cell layer maybe become one of the solving methods. In this study, we prepared the PVA nanofiber mat by using the electrospinning method and the surface modification of the PVA nanofiber was studied to improve the durability of the corneal epithelium layer. The collagen-immobilized PVA nanofiber sheets could support the adhesion and proliferation of rabbit corneal epithelial cells. And the stratified corneal epithelium structure was observed on the PVA nanofiber sheets when the epithelium was co-cultured with rabbit corneal stromal cells. It means that the corneal epithelium was well differentiated on the collagen immobilized PVA nanofiber sheet. The stability of the corneal epithelium layer on the PVA was dramatically improved; the stratified epithelium layer was kept for two weeks after the differentiation introduction, totally after one month. A light transmittance of these materials is not yet enough. Further study to improve the transmission of light, is required.

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Development and Validation of Fluorescently Labeled, Functional Type I Collagen Molecules

10.1101/2021.03.26.437209 ◽

2021 ◽

Author(s):

Seyed Mohammad Siadat ◽

Monica E Susilo ◽

Jeffrey A Paten ◽

Alexandra A Silverman ◽

Charles A DiMarzio ◽

...

Keyword(s):

Type I Collagen ◽

De Novo ◽

Type I ◽

Growth And Remodeling ◽

Fibril Structure ◽

Collagen Molecules ◽

Vitro Model ◽

Development And Validation ◽

Network Morphology

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are quantified for the first time. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ~1.2 labels preserves them. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labelled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development, is endocytosed rapidly by cells and is translocated into synthesized matrix networks within 24 hours.

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Anti-Photoaging Effect of Plant Extract Fermented with Lactobacillus buchneri on CCD-986sk Fibroblasts and HaCaT Keratinocytes

Journal of Functional Biomaterials ◽

10.3390/jfb11010003 ◽

2020 ◽

Vol 11 (1) ◽

pp. 3 ◽

Cited By ~ 2

Author(s):

Yun-Mi Kang ◽

Chul-Hee Hong ◽

Sa-Haeng Kang ◽

Dong-Seok Seo ◽

Seong-Oh Kim ◽

...

Keyword(s):

In Vitro Model ◽

Type I Collagen ◽

Dermal Fibroblasts ◽

Epidermal Keratinocytes ◽

Type I ◽

Mrna Levels ◽

Protective Effects ◽

Lactobacillus Buchneri ◽

Vitro Model

Ultraviolet (UV) exposure triggers the abnormal production of reactive oxygen (ROS) species and the expression of matrix metalloproteinases (MMPs) that are responsible for photoaging. Probiotics are widely used in healthcare and for immune enhancement. One probiotic, Lactobacillus buchneri is found in Kimchi. This study was aimed at assessing the anti-photoaging effect of plant extracts fermented with L. buchneri (PELB) to develop functional cosmetics. We investigated the anti-photoaging effect of PELB in a UVB-induced photoaging in vitro model and selected effective extracts using the elastase inhibition assay, ELISA for Type I procollagen and collagenase-1, and quantitative real time PCR. Normal human dermal fibroblasts and epidermal keratinocytes were pre-treated with PELB and exposed to UVB. We found that PELB decreased elastase activity and increased type I collagen expression in a UVB-induced photoaging in vitro model. In addition, PELB greatly reduced collagenase activity and MMP mRNA levels in a UVB-induced photoaging in vitro model. Furthermore, PELB promoted the expression of moisture factor and anti-oxidant enzymes in a UVB-induced photoaging in vitro model. These results indicated that the PELB could be potential candidates for the protective effects against UVB-induced photoaging. Overall, these results suggest that PELB might be useful natural components of cosmetic products.

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A serum-free in vitro model of renal microvessel development

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.6.f1150 ◽

1998 ◽

Vol 274 (6) ◽

pp. F1150-F1160 ◽

Cited By ~ 2

Author(s):

Lisa M. Antes ◽

Monica M. Villar ◽

Sylvia Decker ◽

Roberto F. Nicosia ◽

Dean A. Kujubu

Keyword(s):

Growth Factors ◽

Endothelial Cell ◽

In Vitro Model ◽

Type I Collagen ◽

Type I ◽

Renal Vasculature ◽

Serum Free ◽

Polypeptide Growth Factors ◽

Vitro Model

The differentiation and organization of the embryonic renal vasculature is a crucial event in renal development. To study this process, we developed a serum-free in vitro model of renal microvessel development. Mouse embryonic kidney explants, when embedded specifically in type I collagen, demonstrate outgrowth of microvascular structures when stimulated by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA, 10–50 ng/ml). Other polypeptide growth factors stimulated little, if any, microvessel outgrowth from the explants. Similar outgrowths were not observed when other embryonic tissue explants were used. The number of microvessels observed depended on the gestational age of the explants. We hypothesize that TPA induces the in situ differentiation of metanephric mesenchymal cells into endothelial cell precursors and that specific matrix proteins and cell-matrix interactions are necessary for the organization of these precursors into microvessels. Our model will allow us to examine in detail the responsiveness of metanephric kidney cells to both growth factors and extracellular matrix molecules and to understand how they influence renal endothelial cell differentiation.

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In Vitro Model of Bartonella henselae-Induced Angiogenesis

Infection and Immunity ◽

10.1128/iai.72.12.7315-7317.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 7315-7317 ◽

Cited By ~ 19

Author(s):

James E. Kirby

Keyword(s):

In Vitro Model ◽

Type I Collagen ◽

Bartonella Henselae ◽

Collagen Matrix ◽

In Vitro Models ◽

Type I ◽

Blood Vessel Formation ◽

Vitro Model

ABSTRACT Bartonella henselae is a gram-negative pathogen that causes angiogenesis. Here, I establish in vitro models to study Bartonella-induced blood vessel formation. I found that B. henselae induces long-term endothelial survival and tubular differentiation within type I collagen matrix.

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Pseudopterosin and O-Methyltylophorinidine Suppress Cell Growth in a 3D Spheroid Co-Culture Model of Pancreatic Ductal Adenocarcinoma

Bioengineering ◽

10.3390/bioengineering7020057 ◽

2020 ◽

Vol 7 (2) ◽

pp. 57

Author(s):

Bailu Xie ◽

Jan Hänsel ◽

Vanessa Mundorf ◽

Janina Betz ◽

Irene Reimche ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

In Vitro Model ◽

Type I Collagen ◽

Ductal Adenocarcinoma ◽

Pancreatic Tumors ◽

Type I ◽

Culture In Vitro ◽

Drug Candidates ◽

Vitro Model

Current therapies for treating pancreatic ductal adenocarcinoma (PDAC) are largely ineffective, with the desmoplastic environment established within these tumors being considered a central issue. We established a 3D spheroid co-culture in vitro model using a PDAC cell line (either PANC-1 or Capan-2), combined with stellate cells freshly isolated from pancreatic tumors (PSC) or hepatic lesions (HSC), and human type I collagen to analyze the efficiency of the chemotherapeutic gemcitabine (GEM) as well as two novel drug candidates derived from natural products: pseudopterosin (PsA-D) and O-methyltylophorinidine (TYLO). Traditional 2D in vitro testing of these agents for cytotoxicity on PANC-1 demonstrated IC50 values of 4.6 (±0.47) nM, 34.02 (±1.35) µM, and 1.99 (±0.13) µM for Tylo, PsA-D, and GEM, respectively; these values were comparable for Capan-2: 5.58 (±1.74) nM, 33.94 (±1.02) µM, and 0.41 (±0.06) µM for Tylo, PsA-D, and GEM, respectively. Importantly, by assessing the extent of viable cells within 3D co-culture spheroids of PANC-1 with PSC or HSC, we could demonstrate a significant lack of efficacy for GEM, while TYLO remained active and PsA-D showed slightly reduced efficacy: GEM in PANC-1/PSC (IC50 = >100 µM) or PANC-1/HSC (IC50 = >100 µM) spheroids, TYLO in PANC-1/PSC (IC50 = 3.57 ± 1.30 nM) or PANC-1/HSC (IC50 = 6.39 ± 2.28 nM) spheroids, and to PsA-D in PANC-1/PSC (IC50 = 54.42 ± 12.79 µM) or PANC-1/HSC (IC50 = 51.75 ± 0.60 µM). Microscopic 3D rendering supported these cytotoxicity outcomes, showing little or no morphological spheroid structure change during this period of rapid cell death. Our results support the use of this 3D spheroid co-culture in vitro model having a desmoplastic microenvironment for the identification of possible novel chemotherapeutic drug candidates for PDAC, such as TYLO and PsA-D.

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