Synthesis and turnover of collagen precursors in rabbit skin.
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ABSTRACT:
SUBMITTER: Nimni ME
PROVIDER: S-EPMC1270220 | biostudies-other | 1967 Jan
REPOSITORIES: biostudies-other
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Project description:Growing rabbits were infused for up to 10 h with labelled proline, tyrosine and leucine to achieve plateau conditions within body free pools, for [3H]proline infusion, blood free-proline specific radioactivity remained constant after about 1 h. For individual animals, type-I- and type-III-collagen precursors were isolated by precipitation with (NH4)2SO4 and DEAE-cellulose chromatography. Experiments where 3H- and 14C-labelled proline and tyrosine were infused concurrently for different periods of time showed that type I procollagen reached plateau specific radioactivity within 3 h and 90% of the plateau value after 2 h infusion, corresponding to a calculated apparent t 1/2 of less than 26 min. Plateau values for type I procollagen were taken as precursor amino acid pool specific radioactivities. The type-III-collagen-precursor fractions consistently showed lower rates of label incorporation and, by assuming that both type I and type III collagens are synthesized from the same amino acid pools, kinetic analysis revealed an apparent t 1/2 for the isolated type-III-collagen precursors of 3.9 h. For proline, there were large variations between animals in the ratio between the precursor pool for collagen synthesis and the skin homogenate free pool (0.31 +/- 0.13, mean +/- S.D.), so that collagen-synthesis rates based solely on total tissue free-pool values for proline are subject to large and inconsistent errors.
| S-EPMC1161127 | biostudies-other
Project description:Renal fibrosis is a hallmark of chronic kidney disease (CKD) caused by an imbalance between formation and degradation of extracellular matrix proteins. We investigated the collagen turnover profile of 81 non-dialysis CKD stage 2-5 patients by measuring peptides reflecting formation and degradation of collagen type (COL) I, III, IV, and VI. Based on the collagen turnover profile, we identified four clusters of patients. Cluster 1 contained one patient with prostate cancer, who had a distinct collagen turnover. The other clusters generally had severe (Cluster 2), moderate (Cluster 4), or mild CKD (Cluster 3). Cluster 4 patients were characterized by higher levels of COL III, COL IV, and COL VI (all p < 0.001) degradation fragments in plasma, while patients in Clusters 2 and 4 had higher levels of COL VI formation (p < 0.05). COL IV fragments in plasma were lower in Cluster 2 (p < 0.01). Urinary COL III fragments decreased from Cluster 3 to 4, and from Cluster 4 to 2 (both p < 0.001). We show that patients with similar kidney function have a different collagen remodeling profile, suggesting that different phenotypes exist with different disease activity and potentially disease progression. Biomarkers of collagen remodeling could provide additional information to traditional markers of renal function.
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Project description:A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.
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Project description:IntroductionPeroxisome proliferator-activated receptor (PPAR) agonists are known to modulate the synthesis of dermal lipids and proteins including collagens. Olive (Olea europaea) leaves have been reported to contain PPAR-binding ligands. Collagen IV, a major dermal-epidermal junction (DEJ) protein, degrades with both age and disease. Here, we report the formulation of a novel multi-ligand complex, Linefade, and its effects on collagen IV synthesis.MethodsLinefade prepared from the leaves of Olea europaea contains 2% w/w plant extract solids dissolved in a mixture of glyceryl monoricinoleate and dimethyl isosorbide. In silico docking was performed with PPAR-α (PDB ID: 2P54). Linefade was evaluated for PPAR-α-dependent transcription in a luciferase reporter assay system. Cell viability and collagen IV levels in human dermal fibroblast cultures were measured using the MTT method and ELISA assay, respectively. Transcriptome analysis was conducted on a full-thickness reconstituted human skin (EpiDermFT) model. Ex vivo cell viability and collagen IV immunostaining were performed on human skin explants.ResultsIn silico docking model of the major constituents (oleanolic acid and glyceryl monoricinoleate) produced a co-binding affinity of -6.7 Kcal/mole. Linefade significantly increased PPAR-α transcriptional activity in CHO cells and collagen IV synthesis in adult human dermal fibroblasts. Transcriptome analysis revealed that 1% Linefade modulated the expression of 280 genes with some related to epidermal differentiation, DEJ, PPAR, Nrf2 and retinoid pathways. An ex vivo human explant study showed that 1% Linefade, delivered via a triglycerides excipient, increased collagen IV levels along the dermal-epidermal junction by 52%.ConclusionIn silico modelling and in vitro and ex vivo analyses confirmed Linefade-mediated activation of PPAR-α and stimulation of collagen IV synthesis.
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Project description:Although aging represents the most important epidemiologic risk factor for fibrotic disease, the reasons for this are incompletely understood. Excess collagen deposition in tissues is the sine qua non of tissue fibrosis and can be viewed as an imbalance between collagen production and collagen degradation. Yet we still lack a detailed understanding of the changes that take place during development, maturation, and aging in extracellular matrix (ECM) dynamics. Resolution of fibrosis is impaired in aging, and this impairment may explain why age is the most important risk factor for fibrotic diseases, such as idiopathic pulmonary fibrosis. However, ECM dynamics and impaired resolution of fibrosis in aging remain understudied. Here we show that cell-mediated collagen uptake and degradation are diminished in aged animals and this finding correlates with downregulation of the collagen endocytic receptor mannose receptor, C-type 2 (Mrc2). We identify myeloid zinc finger-1 as a potentially novel transcriptional regulator of Mrc2, and both this transcription factor and Mrc2 are downregulated in multiple tissues and organisms in an age-dependent manner. Thus, cell-mediated degradation of collagen is an essential process that promotes resolution of fibrosis, and impairment in this process contributes to age-related fibrosis.
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Project description:Gene therapies have great potential in regenerative medicine; however, clinical translation has been inhibited by low stability and limited transfection efficiencies. Herein, we incorporate collagen-mimetic peptide (CMP)-linked polyplexes in collagen scaffolds to increase DNA stability by up to 400% and enable tailorable in vivo transgene expression at 100-fold higher levels and 10-fold longer time periods. These improvements were directly linked to a sustained interaction between collagen and polyplexes that persisted during cellular remodeling, polyplex uptake, and intracellular trafficking. Specifically, incorporation of CMPs into polyethylenimine (PEI) polyplexes preserved serum-exposed polyplex-collagen activity over a period of 14days, with 4 orders-of-magnitude more intact DNA present in CMP-modified polyplex-collagen relative to unmodified polyplex-collagen after a 10day incubation under cell culture conditions. CMP-modification also altered endocytic uptake, as indicated by gene silencing studies showing a nearly 50% decrease in transgene expression in response to caveolin-1 silencing in modified samples versus only 30% in unmodified samples. Furthermore, cellular internalization studies demonstrated that polyplex-collagen association persisted within cells in CMP polyplexes, but not in unmodified polyplexes, suggesting that CMP linkage to collagen regulates intracellular transport. Moreover, experiments in an in vivo repair model showed that CMP modification enabled tailoring of transgene expression from 4 to 25days over a range of concentrations. Overall, these findings demonstrate that CMP decoration provides substantial improvements in gene retention, altered release kinetics, improved serum-stability, and improved gene activity in vivo. This versatile technique has great potential for multiple applications in regenerative medicine.Statement of significanceIn this work, we demonstrate a novel approach for stably integrating DNA into collagen scaffolds to exploit the natural process of collagen remodelling for high efficiency non-viral gene delivery. The incorporation of CMPs into DNA polyplexes, coupled with the innate affinity between CMPs and collagen, not only permitted improved control over polyplex retention and release, but also provided a series of substantial and highly unique benefits via the stable and persistent linkage between CMP-polyplexes and collagen fragments. Specifically, CMP-modification of polyplexes was demonstrated to (i) control release for nearly a month, (ii) improve vector stability under physiological-like conditions, and (iii) provide ligands able to efficiently transfer genes via endocytic collagen pathways. These unique properties overcome key barriers inhibiting non-viral gene therapy.
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Project description:Enzymically isolated rabbit aortic smooth-muscle cells (SMC) in the first few days of primary culture express a 'contractile phenotype', but with time these cells modulate to a 'synthetic phenotype'. Synthetic-state SMC are able to proliferate, and, provided that they undergo fewer than 5 cumulative population doublings, return to the contractile phenotype after reaching confluency [Campbell, Kocher, Skalli, Gabbiani & Campbell (1989) Arteriosclerosis 9, 633-643]. The present study has determined the synthesis of collagen, at the protein and mRNA levels, by cultured SMC as they undergo a change in phenotypic state. The results show that, upon modulating to the synthetic phenotype, SMC synthesized 25-30 times more collagen than did contractile cells. At the same time, non-collagen-protein synthesis increased only 5-6-fold, indicating a specific stimulation of collagen synthesis. Steady-state mRNA levels are also elevated, with alpha 2(I) and alpha 1(III) mRNA levels 30 times and 20 times higher respectively, probably reflecting increased transcriptional activity. Phenotypic modulation was also associated with an alteration in the relative proportions of type I and III collagens synthesized, contractile SMC synthesizing 78.1 +/- 3.6% (mean +/- S.D.) type I collagen and 17.5 +/- 4.7% type III collagen, and synthetic cells synthesizing 90.3 +/- 2.0% type I collagen and 5.8% +/- 1.8% type III collagen. Enrichment of type I collagen was similarly noted at the mRNA level. On return to the contractile state, at confluency, collagen production and the percentage of type I collagen decreased. This further illustrates the close association between the phenotypic state of SMC and their collagen-biosynthetic phenotype.
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Project description:In white adipose tissue, adipocytes and adipocyte precursor cells are enmeshed in a dense network of type I collagen fibrils. The fate of this pericellular collagenous web in diet-induced obesity, however, is unknown. This study seeks to identify the genetic underpinnings of proteolytic collagen turnover and their association with obesity progression in mice and humans.The hydrolysis and degradation of type I collagen at early stages of high-fat diet feeding was assessed in wild-type or MMP14 (MT1-MMP)-haploinsufficient mice using immunofluorescent staining and scanning electron microscopy. The impact of MMP14-dependent collagenolysis on adipose tissue function was interrogated by transcriptome profiling with cDNA microarrays. Genetic associations between MMP14 gene common variants and obesity or diabetes traits were examined in a Japanese cohort (n = 3,653).In adult mice, type I collagen fibers were cleaved rapidly in situ during a high-fat diet challenge. By contrast, in MMP14 haploinsufficient mice, animals placed on a high-fat diet were unable to remodel fat pad collagen architecture and display blunted weight gain. Moreover, transcriptional programs linking type I collagen turnover with adipogenesis or lipogenesis were disrupted by the associated decrease in collagen turnover. Consistent with a key role played by MMP14 in regulating high-fat diet-induced metabolic programs, human MMP14 gene polymorphisms located in proximity to the enzyme's catalytic domain were closely associated with human obesity and diabetes traits.Together, these findings demonstrate that the MMP14 gene, encoding the dominant pericellular collagenase operative in vivo, directs obesogenic collagen turnover and is linked to human obesity traits.
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