scholarly journals Inhibition of prolyl hydroxylation and procollagen processing in chick-embryo calvaria by a derivative of pyridine-2,4-dicarboxylate. Characterization of the diethyl ester as a proinhibitor

1991 ◽  
Vol 275 (2) ◽  
pp. 469-476 ◽  
Author(s):  
G Tschank ◽  
D G Brocks ◽  
K Engelbart ◽  
J Mohr ◽  
E Baader ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Type I Collagen ◽  
Helical Structure ◽  
Diethyl Ester ◽  
Type I ◽  
Dependent Manner ◽  
Maximal Inhibition ◽  
Collagen Formation ◽  
Collagenous Material

The biochemical and morphological consequences of procollagen prolyl 4-hydroxylase inhibition by pyridine-2,4-dicarboxylic acid (2,4-PDCA) and its diethyl ester (diethyl-2,4-PDC) were studied in chick-embryo calvaria, which predominantly synthesize type I collagen. Half-maximal inhibition of tissue hydroxyproline formation required 650 microM-2,4-PDCA, whereas the Ki with respect to chicken prolyl 4-hydroxylase in vitro was 2 microM. In contrast, half-maximal inhibition was caused by 10 microM-diethyl-2,4-PDC in the intact calvaria, although chicken prolyl 4-hydroxylase in vitro was not inhibited even at 1 mM. The collagenous material produced in the presence of diethyl-2,4-PDC showed an altered ‘melting’ profile and a lowering of the transition temperature by 10 degrees C, indicating misalignment and thermal instability of its triple-helical structure. Amount and electrophoretic mobility of procollagen type I chains were increased in a dose-dependent manner. The amounts of partially processed species and alpha-chains were decreased, without change in mobility. This marked effect on procollagen-collagen conversion in the intact calvaria suggests that the underhydroxylated collagenous material generated in the presence of diethyl-2,4-PDC is resistant to or acts as endogenous secondary inhibitor of type I procollagen N-proteinase. Electron microscopy of treated calvaria cells showed dilated rough endoplasmic reticulum and numerous phagolysosomes, indicating intracellular retention and lysosomal degradation of the newly synthesized underhydroxylated collagenous material. In summary, these results identify 2,4-PDCA and diethyl-2,4-PDC as the first prolyl 4-hydroxylase-directed inhibitor/proinhibitor pair that affects intra- and extra-cellular events during collagen formation.

scholarly journals Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta

2021 ◽  
Vol 10 (14) ◽  
pp. 3141
Author(s):  
Hyerin Jung ◽  
Yeri Alice Rim ◽  
Narae Park ◽  
Yoojun Nam ◽  
Ji Hyeon Ju
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Disease Modeling ◽  
Bone Fragility ◽  
Osteogenic Potential ◽  
Type I ◽  
Gene Correction ◽  
Col1a1 Gene ◽  
Collagen Formation

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

scholarly journals SAT0298 IS INTERLEUKIN 6 A FACTOR OF FIBROGENESIS IN DERMAL FIBROBLASTS?

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1094.1-1094
Author(s):  
A. S. Siebuhr ◽  
P. Juhl ◽  
M. Karsdal ◽  
A. C. Bay-Jensen
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Collagen Type ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Full Time ◽  
Collagen Formation ◽  
Type Iii

Background:Interleukin 6 (IL-6) is known to have both pro- and anti-inflammatory properties, depending on the receptor activation. The classical IL-6 signaling via the membrane bound receptor is mainly anti-inflammatory, whereas signaling through the soluble receptor (sIL-6R) is pro-inflammatory/pro-fibrotic. However, the direct fibrotic effect of IL-6 stimulation on dermal fibroblasts is unknown.Objectives:We investigated the fibrotic effect of IL-6 + sIL-6R in a dermal fibroblast model and assessed fibrosis by neo-epitope biomarkers of extracellular matrix proteins.Methods:Primary healthy human dermal fibroblasts were grown for up to 17 days in DMEM medium with 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid. IL-6 [1-90 nM]+sIL-6R [0.1-9 nM] alone or in combination with TGFβ [1 nM] were tested in three different donors. TGFβ [1 nM], PDGF-AB [3 nM] and non-stimulated cells (w/o) were used as controls. Tocilizumab (TCZ) with TGFβ + IL-6 + sIL-6R stimulation was tested in one donor. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Western blot analysis investigated signal cascades. Gene expression of selected ECM proteins was analyzed. Statistical analyses included One-way and 2-way ANOVA and area under the curve analysis.Results:formation by the end of the culture period. The fibronectin and collagen type VI signal were consistent between the three tested donors, whereas the formation of type III collagen was only increased in one donor, but in several trials. Type I collagen formation was unchanged by IL-6 + sIL-6R stimulation. The gene expression of type I collagen was induced by IL-6 + sIL-6R. Western blot analysis validated trans-signaling by the IL-6+sIL-6R stimulation as expected.IL-6 + sIL-6R stimulation in combination with TGFβ decreased fibronectin levels compared to TGFβ alone but did not reach the level of unstimulated fibroblasts. The formation of collagen type IV was generally unchanged with IL-6 + sIL-6R + TGFβ compared to TGFβ alone. Collagen type I and III formation was more scattered in the signals when IL-6 + sIL-6R was in combination with TGFβ, as the biomarker level could be either decreased or increased compared to TGFβ alone. In two studies the type I collagen level was synergistic increased by IL-6 + sIL-6R + TGFβ, whereas another study found the level to be decreased compared to TGFβ alone. The gene expression of fibronectin and type I collagen was increased with TGFβ +IL-6+sIL-6R compared to TGFβ alone.Inhibition of IL-6R by TCZ in combination with IL-6 + sIL-6R did only decrease the fibronectin level with the lowest TCZ concentration (p=0.03). TCZ alone decreased the fibronectin level in a dose-dependent manner (One-way ANOVA p=0.0002).Conclusion:We investigated the fibrotic response of dermal fibroblasts to IL-6 + sIL-6R stimulation. IL-6 modulated the fibronectin level and modulated the collagen type III formation level in a somewhat dose-dependent manner. In combination with TGFβ, IL-6 decreased collagen type I and IV formation and fibronectin. However, in this study inhibition of IL-6R by TCZ did not change the fibrotic response of the dermal fibroblasts. This study indicated that IL-6 did not induce collagen formation in dermal fibroblasts, except type III collagen formation with high IL-6 concentration.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Pernille Juhl Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.

Bradykinin augments fibroblast-mediated contraction of released collagen gels

2001 ◽  
Vol 281 (1) ◽  
pp. L164-L171 ◽  
Author(s):  
Tadashi Mio ◽  
Xiangde Liu ◽  
Myron L. Toews ◽  
Yuichi Adachi ◽  
Debra J. Romberger ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Type I Collagen ◽  
Inflammatory Diseases ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Collagen Gels ◽  
Protein Kinase C Inhibitors

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10−9 and 10−8 M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.

Bone Morphogenetic Protein-2 Inhibits Differentiation and Mineralization of Cementoblasts in vitro

2003 ◽  
Vol 82 (1) ◽  
pp. 23-27 ◽  
Author(s):  
M. Zhao ◽  
J.E. Berry ◽  
M.J. Somerman
Keyword(s):  
Bone Morphogenetic Protein ◽  
Type I Collagen ◽  
Bone Morphogenetic Protein 2 ◽  
Nodule Formation ◽  
Type I ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Morphogenetic Protein ◽  
Regenerative Therapies

As an approach for improving the outcome and predictability of periodontal regenerative therapies, we have focused on determining the responses of cells within the local environment to putative regenerative factors. This study examined the effects of bone morphogenetic protein-2 (BMP-2) on murine cementoblasts in vitro. Northern blot analysis indicated that BMP-2 decreased mRNA levels of bone sialoprotein and type I collagen dose-dependently (10–300 ng/mL). At low doses, up to 100 ng/mL, BMP-2 had no effect on transcripts for osteocalcin and osteopontin, whereas at 300 ng/mL, BMP-2 greatly increased expression of these two genes. BMP-2 also inhibited cementoblast-mediated mineral nodule formation in a dose-dependent manner (inhibition was noted at 10 ng/mL). Noggin reversed the effects of BMP-2 on gene expression and on mineralization. These findings reflect the diverse responses of periodontal cells to BMP-2 and highlight the need to consider the complexity of factors involved in designing predictable regenerative therapies.

High dickkopf-1 levels in sera and leukocytes from children with 21-hydroxylase deficiency on chronic glucocorticoid treatment

2013 ◽  
Vol 304 (5) ◽  
pp. E546-E554 ◽  
Author(s):  
Giacomina Brunetti ◽  
Maria Felicia Faienza ◽  
Laura Piacente ◽  
Annamaria Ventura ◽  
Angela Oranger ◽  
...  
Keyword(s):  
Type I Collagen ◽  
High Serum ◽  
Type I ◽  
Dependent Manner ◽  
Hydroxylase Deficiency ◽  
Drug Induced ◽  
Glucocorticoid Treatment ◽  
21 Hydroxylase Deficiency ◽  
Dickkopf 1

Children with 21-hydroxylase deficiency (21-OHD) need chronic glucocorticoid (cGC) therapy to replace congenital deficit of cortisol synthesis, and this therapy is the most frequent and severe form of drug-induced osteoporosis. In this study, we enrolled 18 patients (9 females) and 18 sex- and age-matched controls. We found in 21-OHD patients high serum and leukocyte levels of dickkopf-1 (DKK1), a secreted antagonist of the Wnt/β-catenin signaling pathway known to be a key regulator of bone mass. In particular, we demonstrated by flow cytometry, confocal microscopy, and real-time PCR that monocytes, T lymphocytes, and neutrophils from patients expressed high levels of DKK1, which may be related to the cGC therapy. In fact, we showed that dexamethasone treatment markedly induced the expression of DKK1 in a dose- and time-dependent manner in leukocytes. The serum from patients containing elevated levels of DKK1 can directly inhibit in vitro osteoblast differentiation and receptor activator of NF-κB ligand (RANKL) expression. We also found a correlation between both DKK1 and RANKL or COOH-terminal telopeptides of type I collagen (CTX) serum levels in 21-OHD patients on cGC treatment. Our data indicated that DKK1, produced by leukocytes, may contribute to the alteration of bone remodeling in 21-OHD patients on cGC treatment.

ADP Receptor Antagonists and Converting Enzyme Systems Reduce Platelet Deposition onto Collagen

1992 ◽  
Vol 67 (04) ◽  
pp. 461-467 ◽  
Author(s):  
William R Wagner ◽  
Jeffrey A Hubbell
Keyword(s):  
Type I Collagen ◽  
Type I ◽  
Dependent Manner ◽  
Receptor Antagonists ◽  
Near Surface ◽  
Enzyme Systems ◽  
Platelet Deposition ◽  
Adp Receptor ◽  
Adp Receptor Antagonists

SummaryThe effect of the ADP receptor antagonists ATP and adenosine 5’-(β, γ-methylene)triphosphate (AMP-PCP), and the ADP-utilizing enzyme systems creatine phosphokinase/creatine phosphate (CPK/CP) and pyruvate kinase/phosphoenol pyruvate (PK/PEP) on platelet deposition onto type I collagen was examined. An in vitro perfusion system was used, which allowed continuous visualization of the deposition of fluorescently labelled platelets. This system also provided well-controlled rheology, precise quantification of deposition, and allowed the use of heparinized whole human blood (3 u/ml). Heparinization at this level permits the local generation of thrombin near surface platelet aggregates. The contribution of ADP is thus studied with the combined effects of thrombin, thromboxane A2, and other aggregating agents present. Results from these studies indicate that ATP was capable of inhibiting deposition by 60% at 1 pM and 90% at 5 μM (whole blood cone.). AMP-PCP inhibited deposition in a dose dependent manner with a Ki of approximately 80 μM and a maximum inhibition of 60%. Inhibition by CPK/CP was measured at 20, 40, and 60 u/ml, with approximately 45% inhibition achieved for the latter two concentrations. PK/ PEP at 60 u/ml resulted in 70% inhibition. These results support a role for ADP in mediating platelet recruitment in thrombus growth on collagen. Previous work utilizing animal bleeding times supports this conclusion; the present study demonstrates that this role is not dependent upon endothelial or vasoconstrictive effects. Intraplatelet cAMP levels were raised with respect to controls upon exposure to ATP at 8.3 μM (p <0.025), and 15 μM (p <0.005), as well as AMP-PCP at 42-500 μM (p <0.005). Since increasing intraplatelet cAMP may affect platelet function, these results raise another possible interpretation for some of our platelet deposition data as well as that of previous experimenters. However, since ATP at 1 μM was not shown to affect intraplatelet cAMP, it is believed that at least part of the ATP-mediated inhibition was due to ADP receptor competition.

Microvessel formation from mouse embryonic aortic explants is oxygen and VEGF dependent

2002 ◽  
Vol 283 (2) ◽  
pp. R487-R495 ◽  
Author(s):  
Tetsu Akimoto ◽  
Helen Liapis ◽  
Marc R. Hammerman
Keyword(s):  
Type I Collagen ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Thoracic Level ◽  
Aortic Explants ◽  
Endothelial Growth Factor

To delineate the roles of O2 and vascular endothelial growth factor (VEGF) in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O2, but not room air, the addition of VEGF to explants stimulated the formation of CD31-positive, Flk-1-positive, Gs-IB4-positive structures in a concentration-dependent manner. Electron microscopy showed the structures to be capillary-like. VEGF-induced capillary-like structure formation was inhibited by sequestration of VEGF via addition of soluble Flt-1 fusion protein or anti-VEGF antibodies. Expression of Flk-1, but not Flt-1, was increased in embryonic aorta cultured under 5% O2 relative to room air. Our data suggest that low O2 upregulates Flk-1 expression in embryonic aorta in vitro and renders it more responsive to VEGF.

Control of type I collagen formation in the lung

1991 ◽  
Vol 261 (2) ◽  
pp. L29-L40 ◽  
Author(s):  
R. H. Goldstein
Keyword(s):  
Type I Collagen ◽  
Potential Interaction ◽  
Complex Nature ◽  
Type I ◽  
Mrna Levels ◽  
Structural Protein ◽  
Inflammatory Reactions ◽  
Collagen Formation ◽  
Collagen Accumulation

Type I collagen is a major structural protein in the lung, the accumulation of which is stimulated during certain inflammatory reactions in the lung. Accumulating evidence suggests that type I collagen formation parallels changes in steady-state mRNA levels. Specific inflammatory substances modulate transcription of collagen genes and stabilization of collagen mRNA in vitro. However, the precise role for any particular mediator during fibrotic processes is difficult to identify because of the complex nature of the inflammatory reaction and potential interaction among mediators. The signal transduction mechanisms that regulate collagen accumulation remain to be defined. This review focuses on the regulation of collagen accumulation in the lung by specific inflammatory substances.

Fibroblast growth factor 2 promotes microvessel formation from mouse embryonic aorta

2003 ◽  
Vol 284 (2) ◽  
pp. C371-C377 ◽  
Author(s):  
Tetsu Akimoto ◽  
Marc R. Hammerman
Keyword(s):  
Type I Collagen ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Type I ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Low Oxygen ◽  
Concentration Dependent Manner ◽  
Fgf Receptor

To delineate the roles that oxygen and fibroblast growth factors (FGFs) play in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O2, but not room air, the addition of FGF2 to explants stimulated the formation of Gs-IB4-positive, CD31-positive, and Flk-1-positive microvessels in a concentration-dependent manner. FGF2-stimulated microvessel formation was inhibited by sequestration of FGF2 via addition of soluble FGF receptor (FGFR) chimera protein or anti-FGF2 antibodies. FGFR1 and FGFR2 were present on explants. Levels of FGFR1, but not FGFR2, were increased in embryonic aorta cultured under 5% O2 relative to room air. Our data suggest that low oxygen upregulates FGFR1 expression in embryonic aorta in vitro and renders it more responsive to FGF2.

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

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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