The Carboxylpropeptide of Type I Procollagen in Skin Fibrillogenesis

Previous studies suggested that the antinopropeptide of type 1 procollagen may initiate fibril formation. The purpose of this investigation was to study the location of the carboxypropeptide of type 1 procollagen during collagen fibrillogenesis. Chick embryonic and posthatching skin specimens were studied by immunofluorescence and immunoelectron microscopy and by immunoblotting with antibodies against the amino and carboxypropeptide of type 1 procollagen. The carboxylpropeptide was demonstrated at the surface of collagen fibrils, 20–40nm in diameter (10-day embryos) and in fibrils, 40–65nm (21-day embryos). In addition, the carboxylpropeptide was found at the cell surface and free in the ground substance. The aminopropeptide was only seen in fibrils, 20–30nm in diameter, as previously reported. Ratios of pN-collagen/pC-collagen increased from 16 days embryonic to 3 and 9 days postembryonic skins. This study suggests that both pN-collagen (antinopropeptide plus collagen) arid pC-collagen (carboxylpropetide plus collagen) participate in fibrillogenesis

Pathogenesis of Scleroderma

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65784/1/j.1365-4362.1984.tb05678.x.pd

Exploring hypotheses of the actions of TGF-beta 1 in epidermal wound healing using a 3D computational multiscale model of the human epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-beta 1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-beta 1 literature-derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units ( keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-beta 1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged ( by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-beta 1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-beta 1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing

Agent based modelling helps in understanding the rules by which fibroblasts support keratinocyte colony formation

Background: Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings: A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions: A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine serum

PPAR? Downregulation by TGF in Fibroblast and Impaired Expression and Function in Systemic Sclerosis: A Novel Mechanism for Progressive Fibrogenesis

The nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) is expressed in multiple cell types in addition to adipocytes. Upon its activation by natural ligands such as fatty acids and eicosanoids, or by synthetic agonists such as rosiglitazone, PPAR-γ regulates adipogenesis, glucose uptake and inflammatory responses. Recent studies establish a novel role for PPAR-γ signaling as an endogenous mechanism for regulating transforming growth factor-ß (TGF-ß)- dependent fibrogenesis. Here, we sought to characterize PPAR-γ function in the prototypic fibrosing disorder systemic sclerosis (SSc), and delineate the factors governing PPAR-γ expression. We report that PPAR-γ levels were markedly diminished in skin and lung biopsies from patients with SSc, and in fibroblasts explanted from the lesional skin. In normal fibroblasts, treatment with TGF-ß resulted in a time- and dose-dependent down-regulation of PPAR-γ expression. Inhibition occurred at the transcriptional level and was mediated via canonical Smad signal transduction. Genome-wide expression profiling of SSc skin biopsies revealed a marked attenuation of PPAR-γ levels and transcriptional activity in a subset of patients with diffuse cutaneous SSc, which was correlated with the presence of a ''TGF-ß responsive gene signature'' in these biopsies. Together, these results demonstrate that the expression and function of PPAR-γ are impaired in SSc, and reveal the existence of a reciprocal inhibitory cross-talk between TGF-ß activation and PPAR-γ signaling in the context of fibrogenesis. In light of the potent anti-fibrotic effects attributed to PPAR-γ, these observations lead us to propose that excessive TGF-ß activity in SSc accounts for impaired PPAR-γ function, which in turn contributes to unchecked fibroblast activation and progressive fibrosis. © 2010 Wei et al

The role of SPARC in extracellular matrix assembly

SPARC is a collagen-binding matricellular protein. Expression of SPARC in adult tissues is frequently associated with excessive deposition of collagen and SPARC-null mice fail to generate a robust fibrotic response to a variety of stimuli. This review summarizes recent advancements in the characterization of the binding of SPARC to collagens and describes the results of studies that implicate a function for SPARC in the regulation of the assembly of basal lamina and fibrillar collagen in the ECM. Potential cellular mechanisms that underlie SPARC activity in ECM deposition are also explored

A role for the collagen I/III and MMP-1/-13 genes in primary inguinal hernia?

BACKGROUND: Abnormal collagen metabolism is thought to play an important role in the development of primary inguinal hernia. This is underlined by detection of altered collagen metabolism and structural changes of the tissue in patients with primary inguinal hernia. However, it is still unknown whether these alterations reflect a basic dysfunction of the collagen synthesis, or of collagen degradation. METHODS: In the present study, we analysed type I and type III procollagen messenger ribonucleic acid (mRNA) and MMP-1 and MMP-13 mRNA in cultured fibroblasts from the skin of patients with primary inguinal hernia, and from patients without hernia (controls) by reverse transcription polymerase chain reaction (RT-PCR) and Northern Blot. RESULTS: The results indicated that the ratio of type I to type III procollagen mRNA was decreased in patients with primary hernia, showing significant differences as compared to controls (p = 0.01). This decrease was mainly due to the increase of type III procollagen mRNA. Furthermore, RT-PCR analysis revealed that the expression of MMP-1 mRNA in patients with primary hernia is equivalent to that of controls (p > 0.05). In addition, MMP-13 mRNA is expressed neither in patients with primary hernia nor in controls. CONCLUSION: We concluded that abnormal change of type I and type III collagen mRNAs contribute to the development of primary inguinal hernia, whereas the expressions of MMP-1 and MMP-13 mRNA appears not to be involved in the development of primary inguinal hernia. Thus, the knowledge on the transcriptional regulation of collagen in patients with primary inguinal hernia may help to understand the pathogenesis of primary inguinal hernia, and implies new therapeutic strategies for this disease

Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e1 bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease

Novel haemodynamic structures in the human glomerulus

To investigate human glomerular structure under conditions of physiological perfusion we have analysed fresh and perfusion fixed normal human glomeruli at physiological hydrostatic and oncotic pressures using serial resin section reconstruction, confocal, multiphoton and electron microscope imaging. Afferent and efferent arterioles (21.5±1.2µm and 15.9±1.2µm diameter), recognised from vascular origins, lead into previously undescribed wider regions (43.2±2.8 µm and 38.4±4.9 µm diameter) we have termed vascular chambers (VCs) embedded in the mesangium of the vascular pole. Afferent VC(AVC) volume was 1.6 fold greater than Efferent VC(EVC) volume. From the AVC long non-branching high capacity conduit vessels (n=7) (Con; 15.9±0.7µm diameter) led to the glomerular edge where branching was more frequent. Conduit vessels have fewer podocytes than filtration capillaries. VCs were confirmed in fixed and unfixed specimens with a layer of banded collagen identified in AVC walls by multiphoton and electron microscopy. Thirteen highly branched efferent first order vessels (E1;9.9±0.4µm diam.) converge on the EVC draining into the efferent arteriole (15.9±1.2µm diam.). Banded collagen was scarce around EVC. This previously undescribed branching topology does not conform to the branching of minimum energy expenditure (Murray’s law), suggesting even distribution of pressure/flow to the filtration capillaries is more important than maintaining the minimum work required for blood flow. We propose that AVCs act as plenum manifolds possibly aided by vortical flow in distributing and balancing blood flow/pressure to conduit vessels supplying glomerular lobules. These major adaptations to glomerular capillary structure could regulate haemodynamic pressure and flow in human glomerular capillaries

Development of a Three Dimensional Multiscale Computational Model of the Human Epidermis

Transforming Growth Factor (TGF-β1) is a member of the TGF-beta superfamily ligand-receptor network. and plays a crucial role in tissue regeneration. The extensive in vitro and in vivo experimental literature describing its actions nevertheless describe an apparent paradox in that during re-epithelialisation it acts as proliferation inhibitor for keratinocytes. The majority of biological models focus on certain aspects of TGF-β1 behaviour and no one model provides a comprehensive story of this regulatory factor's action. Accordingly our aim was to develop a computational model to act as a complementary approach to improve our understanding of TGF-β1. In our previous study, an agent-based model of keratinocyte colony formation in 2D culture was developed. In this study this model was extensively developed into a three dimensional multiscale model of the human epidermis which is comprised of three interacting and integrated layers: (1) an agent-based model which captures the biological rules governing the cells in the human epidermis at the cellular level and includes the rules for injury induced emergent behaviours, (2) a COmplex PAthway SImulator (COPASI) model which simulates the expression and signalling of TGF-β1 at the sub-cellular level and (3) a mechanical layer embodied by a numerical physical solver responsible for resolving the forces exerted between cells at the multi-cellular level. The integrated model was initially validated by using it to grow a piece of virtual epidermis in 3D and comparing the in virtuo simulations of keratinocyte behaviour and of TGF-β1 signalling with the extensive research literature describing this key regulatory protein. This research reinforces the idea that computational modelling can be an effective additional tool to aid our understanding of complex systems. In the accompanying paper the model is used to explore hypotheses of the functions of TGF-β1 at the cellular and subcellular level on different keratinocyte populations during epidermal wound healing