Changes in glycation of fibrous type I collagen during long-term in vitro incubation with glucose.
Journal of protein chemistry
The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.
10.1023/b:jopc.0000005501.48719.84
Cell response to matrix mechanics: focus on collagen.
Plant Anne L,Bhadriraju Kiran,Spurlin Tighe A,Elliott John T
Biochimica et biophysica acta
Many model systems and measurement tools have been engineered for observing and quantifying the effect of mechanics on cellular response. These have contributed greatly to our current knowledge of the molecular events by which mechanical cues affect cell biology. Cell responses to the mechanical properties of type 1 collagen gels are discussed, followed by a description of a model system of very thin, mechanically tunable collagen films that evoke similar responses from cells as do gel systems, but have additional advantages. Cell responses to thin films of collagen suggest that at least some of the mechanical cues that cells can respond to in their environment occur at the sub-micron scale. Mechanical properties of thin films of collagen can be tuned without altering integrin engagement, and in some cases without altering topology, making them useful in addressing questions regarding the roles of specific integrins in transducing or mitigating responses to mechanical cues. The temporal response of cells to differences in ECM may provide insight into mechanisms of signal transduction.
10.1016/j.bbamcr.2008.10.012
Immunohistochemical video-microdensitometry of myocardial collagen type I and type III.
Boerrigter G,Mundhenke M,Stark P,Schulte H D,Strauer B E,Schwartzkopff B
The Histochemical journal
Collagen is an essential part of the cardiac interstitium. Collagen subtypes, their location, total amount and the architecture of the fibrillar network are of functional importance. Architecture in terms of density of the fibrillar network is assumed to be reflected by the intensity of immunohistochemical staining of collagen. The aim of this study was to evaluate a video-based microdensitometric method for quantifying density expressed as absorbance of collagen subtypes I and III stained with an indirect immunoperoxidase method in myectomy specimens of patients with hypertrophic obstructive cardiomyopathy. Various factors influencing the immunohistochemical staining product and the technical properties of the image analysis system were investigated. Linearity between collagen concentration and the absorbance of the immunohistochemical staining product was demonstrated for collagen I using a dot-blot technique. Immunohistochemical collagen staining and density measurement were easily reproducible. The cardiac disability of the patients was assessed according to the New York Heart Association (NYHA) criteria. There was a significant increase in collagen type I density with higher NYHA class, whereas no significant association was found for total collagen area fraction. Thus, video-based microdensitometry gives further insight into the structural remodelling of myocardial collagens and reveals their significance in the process of heart failure in hypertrophic cardiomyopathy.
Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta.
Watson Chris J,Phelan Dermot,Collier Patrick,Horgan Stephen,Glezeva Nadia,Cooke Gordon,Xu Maojia,Ledwidge Mark,McDonald Kenneth,Baugh John A
Connective tissue research
Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.
10.3109/03008207.2014.904856
Intracellular interaction of Hsp47 and type I collagen in corneal endothelial cells.
Gu X,Ko M K,Kay E P
Investigative ophthalmology & visual science
PURPOSE:Previous studies by the current investigators showed that type I collagen was posttranslationally regulated in corneal endothelial cells (CECs). These cells synthesize type I procollagen and degrade it intracellularly; however, when CECs are modulated with fibroblast growth factor-2 and/or corneal endothelium modulation factor, they synthesize and secrete type I collagen. Heat shock protein 47 (Hsp47), an endoplasmic reticulum resident protein, is known to function as a molecular chaperon in regulating procollagen folding and/or assembly. The interaction of Hsp47 with type I procollagen synthesis in CECs was also studied. METHODS:Expression of proteins was analyzed by radioactive labeling or immunoblot analysis. The steady state level of type I collagen and Hsp47 mRNAs was determined by northern blot analysis. Coprecipitation using immunoprecipitation followed by immunoblotting was performed to determine the association profile between Hsp47 and type I procollagen. Subcellular localization of Hsp47 and type I procollagen was determined by immunofluorescent staining. RESULTS:Normal and modulated cells expressed Hsp47 and Hsp70. The relative amount of Hsp47 produced by modulated cells was much higher than that of control cells, but the expression level of Hsp70 was the same in control and modulated cells. The steady state levels of type I collagen transcripts were higher in normal cells than in modulated cells, whereas modulated cells contained a much higher steady state level of Hsp47 mRNA. Type I procollagen was found to be associated with Hsp47 when analyzed by coprecipitation or cross-linking. The cytoplasmic localization profile of Hsp47 and type I collagen was different in normal cells, although a colocalization profile was observed to some degree. These two proteins were predominantly colocalized in the Golgi area in modulated CECs. CONCLUSIONS:Hsp47 may be involved in the synthesis and/or intracellular transport of type I collagen in CECs. Modulated cells that secrete type I collagen into the extracellular matrix express a higher level of Hsp47 than do control cells.
The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro.
Schlie-Wolter Sabrina,Ngezahayo Anaclet,Chichkov Boris N
Experimental cell research
Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions-which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance.
10.1016/j.yexcr.2013.03.016
Deletions and duplications of Gly-Xaa-Yaa triplet repeats in the triple helical domains of type I collagen chains disrupt helix formation and result in several types of osteogenesis imperfecta.
Pace J M,Atkinson M,Willing M C,Wallis G,Byers P H
Human mutation
Triple helix formation is a prerequisite for the passage of type I procollagen from the endoplasmic reticulum and secretion from the cell to form extracellular fibrils that will support mineral deposition in bone. Analysis of cDNA from 11 unrelated individuals with osteogenesis imperfecta (OI) revealed the presence of 11 novel, short in-frame deletions or duplications of three, nine, or 18 nucleotides in the helical coding regions of the COL1A1 and COL1A2 collagen genes. Triple helix formation was impaired, type I collagen alpha chains were post-translationally overmodified, and extracellular secretion was markedly reduced. With one exception, the obligate Gly-Xaa-Yaa repeat pattern of amino acids in the helical domains was not altered, but the Xaa- and Yaa position residues were out of register relative to the amino acid sequences of adjacent chains in the triple helix. Thus, the identity of these amino acids, in addition to third position glycines, is important for normal helix formation. These findings expand the known repertoire of uncommon in-frame deletions and duplications in OI, and provide insight into normal collagen biosynthesis and collagen triple helix formation.
10.1002/humu.1193
Collagen as a Biomaterial for Skin and Corneal Wound Healing.
Journal of functional biomaterials
The cornea and the skin are two organs that form the outer barrier of the human body. When either is injured (e.g., from surgery, physical trauma, or chemical burns), wound healing is initiated to restore integrity. Many cells are activated during wound healing. In particular, fibroblasts that are stimulated often transition into repair fibroblasts or myofibroblasts that synthesize extracellular matrix (ECM) components into the wound area. Control of wound ECM deposition is critical, as a disorganized ECM can block restoration of function. One of the most abundant structural proteins in the mammalian ECM is collagen. Collagen type I is the main component in connective tissues. It can be readily obtained and purified, and short analogs have also been developed for tissue engineering applications, including modulating the wound healing response. This review discusses the effect of several current collagen implants on the stimulation of corneal and skin wound healing. These range from collagen sponges and hydrogels to films and membranes.
10.3390/jfb13040249
UV damage of collagen: insights from model collagen peptides.
Biopolymers
Fibrils of Type I collagen in the skin are exposed to ultraviolet (UV) light and there have been claims that collagen photo-degradation leads to wrinkles and may contribute to skin cancers. To understand the effects of UV radiation on collagen, Type I collagen solutions were exposed to the UV-C wavelength of 254 nm for defined lengths of time at 4°C. Circular dichroism (CD) experiments show that irradiation of collagen leads to high loss of triple helical content with a new lower thermal stability peak and SDS-gel electrophoresis indicates breakdown of collagen chains. To better define the effects of UV radiation on the collagen triple-helix, the studies were extended to peptides which model the collagen sequence and conformation. CD studies showed irradiation for days led to lower magnitudes of the triple-helix maximum at 225 nm and lower thermal stabilities for two peptides containing multiple Gly-Pro-Hyp triplets. In contrast, the highest radiation exposure led to little change in the T(m) values of (Gly-Pro-Pro)(10) and (Ala-Hyp-Gly)(10) , although (Gly-Pro-Pro)(10) did show a significant decrease in triple helix intensity. Mass spectroscopy indicated preferential cleavage sites within the peptides, and identification of some of the most susceptible sites of cleavage. The effect of radiation on these well defined peptides gives insight into the sequence and conformational specificity of photo-degradation of collagen.
10.1002/bip.21725
Biomimetic soluble collagen purified from bones.
Ferreira Ana Marina,Gentile Piergiorgio,Sartori Susanna,Pagliano Cristina,Cabrele Chiara,Chiono Valeria,Ciardelli Gianluca
Biotechnology journal
Type I collagen has been extensively exploited as a biomaterial for biomedical applications and drug delivery; however, small molecular alterations occurring during the isolation procedure and its interaction with residual bone extracellular matrix molecules or proteins might affect the overall material biocompatibility and performance. The aim of the current work is to study the potential alterations in collagen properties and organization associated with the absence of proteoglycans, which mimic pathological conditions associated with age-related diseases. A new approach for evaluating the effect of proteoglycans on the properties of isolated type I collagen from the bone matrix is described. Additional treatment with guanidine hydrochloride was introduced to remove residual proteoglycans from the collagen matrix. The properties of the isolated collagen with/without guanidine hydrochloride treatment were investigated and compared with a commercial rabbit collagen as control. We demonstrate that the absence of proteoglycans in the isolated type I collagen affects its thermal properties, the extraction into its native structure, and its ability to hydrate and self-assemble into fibers. The fine control and tuning of all these features, linked to the absence of non-collagenous proteins as proteoglycans, offer the possibility of designing new strategies and biomaterials with advanced biomimetic properties aimed at regenerating bone tissue in the case of fragility and/or defects.
10.1002/biot.201200184
Current Insights into Collagen Type I.
Polymers
Collagen type I (Col-I) is unique due to its high biocompatibility in human tissue. Despite its availability from various sources, Col-I naturally mimics the extracellular matrix (ECM) and generally makes up the larger protein component (90%) in vasculature, skin, tendon bone, and other tissue. The acceptable physicochemical properties of native Col-I further enhance the incorporation of Col-I in various fields, including pharmaceutical, cosmeceutical, regenerative medicine, and clinical. This review aims to discuss Col-I, covering the structure, various sources of availability, native collagen synthesis, current extraction methods, physicochemical characteristics, applications in various fields, and biomarkers. The review is intended to provide specific information on Col-I currently available, going back five years. This is expected to provide a helping hand for researchers who are concerned about any development on collagen-based products particularly for therapeutic fields.
10.3390/polym13162642
Insight into the degradation of type-I collagen fibrils by MMP-8.
Lu Kuojung G,Stultz Collin M
Journal of molecular biology
Although a number of studies have shed light on the mechanism of collagen degradation in solution, the precise mechanism of collagenolysis in the native fibrillar state remains unclear. To gain insight into the mechanism of fibrillar degradation, we calculated the conformational free-energy landscape for unfolding regions of the α2 chain of type-I collagen within the context of the microfibril. Our data suggest that, relatively, imino-rich sequences maintain the canonical triple-helical structure at body temperature. By contrast, the unique MMP (matrix metalloproteinase) cleavage site adopts conformations where the α2 chain is dissociated from the rest of the fibril--behavior that is similar to what was observed in unfolding simulations of isolated collagen-like model peptides in solution. However, the dissociated cleavage site does not fit within the catalytic site of MMP-8, a representative fibrillar collagenase. Additional free-energy simulations suggest that the presence of the catalytic domain leads to a reorientation of the α2 chain such that it adopts a pose that is complementary to the enzyme's active site. These observations argue that, in the fibrillar state, there is a synergy between the normal thermal fluctuations of the substrate when the enzyme is absent and the fluctuations of the substrate when the enzyme is present. More precisely, our findings suggest that thermal fluctuations serve as the driving force for a degradative process that requires both an unfolded cleavage site and the presence of the enzyme.
10.1016/j.jmb.2013.02.002
Evaluation of collagen gel and hyaluronic acid as vitreous substitutes.
Nakagawa M,Tanaka M,Miyata T
Ophthalmic research
We evaluated alkaline-solubilized collagen, hyaluronic acid (HA), and a substance formed from mixing both materials as vitreous substitutes in the rabbit. Fluorescein isothiocyanate (FITC)-labeled collagen (2%), fluorescein-labeled HA (FLHA, 1%), or the mixture was injected into the rabbit vitreous after vitrectomy. After 3 months, histologic study revealed that the substitutes caused no adverse effects on the ocular tissue. Kinetic studies showed that the half-lives of FITC-collagen, FL-HA, and the mixed substance were 5.70, 2.09, and 8.41 days, respectively. The results indicated that HA enhances the collagen half-life. The concomitant of collagen and HA is safe and effective for 3 months in rabbit's eye as a vitreous substitute, and the mixture is capable of tamponade.
10.1159/000268042
Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix.
Kolácná L,Bakesová J,Varga F,Kostáková E,Plánka L,Necas A,Lukás D,Amler E,Pelouch V
Physiological research
ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.
10.33549/physiolres.931302
The materials science of collagen.
Sherman Vincent R,Yang Wen,Meyers Marc A
Journal of the mechanical behavior of biomedical materials
Collagen is the principal biopolymer in the extracellular matrix of both vertebrates and invertebrates. It is produced in specialized cells (fibroblasts) and extracted into the body by a series of intra and extracellular steps. It is prevalent in connective tissues, and the arrangement of collagen determines the mechanical response. In biomineralized materials, its fraction and spatial distribution provide the necessary toughness and anisotropy. We review the structure of collagen, with emphasis on its hierarchical arrangement, and present constitutive equations that describe its mechanical response, classified into three groups: hyperelastic macroscopic models based on strain energy in which strain energy functions are developed; macroscopic mathematical fits with a nonlinear constitutive response; structurally and physically based models where a constitutive equation of a linear elastic material is modified by geometric characteristics. Viscoelasticity is incorporated into the existing constitutive models and the effect of hydration is discussed. We illustrate the importance of collagen with descriptions of its organization and properties in skin, fish scales, and bone, focusing on the findings of our group.
10.1016/j.jmbbm.2015.05.023