An in vitro model to evaluate the properties of matrices produced by fibroblasts from osteogenesis imperfecta and Ehlers-Danlos Syndrome patients

Aim of the studyOsteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts.Materials and methodsFibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled ¹⁴C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content.Results and conclusionsOsteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms.     

Collagenase gene expression in fibroblasts is regulated by a three-dimensional contact with collagen

Collagenase activity in fibroblasts is regulated by cytokines and the interaction with the extracellular matrix. In this study we demonstrate that fibroblasts cultured within a three-dimensional collagen gel show a strong induction of collagenase gene expression. In addition to increased de novo synthesis most of the secreted enzyme was found to be activated leading to a high collagenolytic activity and complete degradation of collagen matrices after removal of fetal calf serum. Collagen I gene expression was found to be reduced under these conditions. These data suggest a specific modulation of cellular metabolism in response to contact with a three-dimensional collagenous matrix resulting in the divergent regulation of collagen and collagenase.     

Involvement of H- and N-Ras isoforms in transforming growth factor-beta1-induced proliferation and in collagen and fibronectin synthesis

Transforming growth factor beta1 (TGF-beta1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-beta and Ras signaling pathways are closely related: TGF-beta1 overcomes Ras mitogenic effects and Ras counteracts TGF-beta signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-beta1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras(-/-)/N-ras(-/-)) isoforms and from heterozygote mice (H-ras(+/-)/N-ras(+/-)). ECM synthesis is increased in basal conditions in H-ras(-/-)/N-ras(-/-) fibroblasts, this increase being higher after stimulation with TGF-beta1. TGF-beta1-induced fibroblast proliferation is smaller in H-ras(-/-)/N-ras(-/-) than in H-ras(+/-)/N-ras(+/-) fibroblasts. Erk activation is decreased in H-ras(-/-)/N-ras(-/-) fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.     

Collagen expression in fibroblasts with a novel LMNA mutation

Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.     

The multifunctional role of fibroblasts during wound healing in Hirudo medicinalis (Annelida, Hirudinea)

Extracellular matrix components play a key role during the angiogenic process for a correct development of blood vessels: fibroblasts are the main cell type involved in the regulation of ECM protein production. In this study we characterize H. medicinalis fibroblasts and demonstrate that they take part to the regulation of angiogenesis that occurs during wound healing process. Massive proliferation and phenotypic modification are two distinctive markers of fibroblast activation. These cells, that are usually responsible for collagen production and function as an energy reservoir, are recruited during wound healing to form a collagen scaffold through a direct mechanic action and through secretion of specific proteoglycans. In addition we show that the activity of fibroblasts is modulated by EGF, a growth factor involved in wound healing in vertebrates. The formation of bundles of collagen fibrils by fibroblasts is fundamental for the development and migration of new blood vessels in lesioned areas during wound repair: administration of lovastatin in explanted leeches affects fibroblasts, damages collagen "scaffold" and indirectly causes the reduction of neo-capillary formation.     

Collagen and fibronectin synthesis by trisomic and triploid fibroblasts from human spontaneous abortuses

Summary Collagen and fibronectin synthesis by trisomic and triploid fibroblasts derived from human spontaneous abortuses was studied. It was demonstrated that the level of fibronectin and collagen production in fibroblasts with trisomy 7, trisomy 9, and triploidy was reduced as compared with diploid cells. A correlation between this observation and an increased rate of intracellular ¹⁴C-procollagen degradation was also established for the anomalous strains. No difference in hydroxylation of ¹⁴C-proline residues in 1() and 2() collagen chains and no fluctuation in the collagen type (): type ratio was found in the strains with the abnormal karyotypes. It was concluded that differentiation of the abnormal fibroblasts was impaired. The data also favour the hypothesis that the deficiency of the fibroblasts in producing proteins may account for a variety of anatomic abnormalities of embryos.     

Adherence,proliferation and collagen turnover by human fibroblasts seeded into different types of collagen sponges

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted non-crosslinked collagen, (2) reconstituted collagen that was chemically crosslinked with either glutaraldehyde, aluminium alginate or acetate, and (3) native collagen fibres, with or without other extracellular matrix molecules (elastin hydrolysate, hyaluronic acid or fibronectin). The non-crosslinked reconstituted collagen was degraded rapidly by human fibroblasts. Teh chemically crosslinked materials proved to be cytotoxic. Native collagen fibres were stable. In the absence of ascorbic acid, the addition of elastin hydrolysate to this type of matrix reduced the rate of collagen degradation. Both elastin hydrolysate and fibronectin partially prevented fibroblast-mediated contraction. Hyaluronic acid was only slightly effective in reducing the collagen degradation rate and more fibroblast-mediated contraction of the material was found than for the native collagen fibres with elastin hydrolysate and fibronectin. In the presence of ascorbate, collagen synthesis was enhanced in the native collagen matrix without additions and in the material containing elastin hydrolysate, but not in the material with hyaluronic acid. These results are indicative of the suitability of tissue substitutes for in vivo application.     

Clonal characterization of fibroblasts in the superficial layer of the adult human dermis

The dermis of adult human skin contains a physiologically heterogeneous population of fibroblasts that interact to produce its unique architecture and that participate in inflammatory and wound repair functions in vivo. This heterogeneity has been well documented for fibroblasts located in the superficial papillary dermis and the deep reticular dermis. However, the existence of diverse fibroblast subpopulations within a given region of the dermis has not been explored. In this study, fibroblast cultures have been established from the superficial dermis following enzymatic dissociation of the tissue. These fibroblasts have been cloned by limiting dilution and initially selected on the basis of morphology and proliferation kinetics. Fibroblasts in some of the clones selected for study express α-smooth muscle actin, a myofibroblast characteristic. Significant differences for fibroblast clones obtained from the same piece of skin have been observed with regard to their rate of collagen lattice contraction, their ability to organize a fibronectin matrix, their release of specific growth factors/cytokines into culture medium, and their response to interleukin-1α. These differences in both morphological and physiological characteristics indicate that the superficial papillary dermis contains a heterogeneous population of fibroblasts. This heterogeneity might indicate that diverse subpopulations of fibroblasts are required to interact in both homeostatic and pathological situations in skin. We thank L’Oréal Life Sciences for providing funding for these studies.     

Use of SILAC for exploring sheddase and matrix degradation of fibroblasts in culture by the PIII SVMP atrolysin A: identification of two novel substrates with functional relevance

Snake venom metalloproteinases (SVMPs) in Viperid venoms primarily function to give rise to local and systemic hemorrhage following snake envenomation. Years of research on these toxins, both in vitro and in vivo, indicate that they function by disrupting capillary basement membranes, stromal matrix and cell-cell and cell-matrix contacts to allow escape of capillary contents under pressure. However, most of these studies used either defined substrates in vitro or were limited by relevant antibodies for detection of sites of action in vivo. In this investigation we use stable isotope-labeled amino acids in culture (SILAC) to determine novel proteolytic activities for exogenously added atrolysin A, a hemorrhagic PIII SVMP isolated from Crotalus atrox venom. When comparing the solubilized products of SILAC-labeled cultured human fibroblasts treated with atrolysin A to that of untreated fibroblasts using LC/MS/MS, several proteins were identified as being released into the culture media specifically due to atrolysin A proteolytic activity. These included collagen VI, fibronectin, fibulin 2 and annexin V. Of particular interest was the observation of collagen VI and annexin V in that the release of these substrates could play a role in altering hemostasis and promote hemorrhage caused by the more typical actions of atrolysin A. In summary, this study demonstrates the utility of SILAC for exploring sheddase activity with cells in culture and suggests the presence of two novel substrates for SVMPs that may play a pathological role in altering host hemostasis during envenomation.     

Comparative proteomic analysis of extracellular matrix proteins secreted by two types of skin fibroblasts

The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.     

RNA interference in human foreskin fibroblasts within the three-dimensional collagen matrix

The technique of RNA interference (RNAi) was trialed in primary human foreskin fibroblasts, both in monolayer culture and in the fibroblast-populated collagen matrix. Knockdown of lamin A/C, p53, and FAK was possible with low-confluency (<50%) monolayer fibroblasts, a transfection vehicle concentration of 1%, and an siRNA concentration of 25–50 nM. Knockdown also was possible in the collagen matrix using similar reagent concentrations and a cellular density of one million fibroblasts per ml of matrix. Optimization of transfection conditions appeared to be important to increase knockdown efficiency. Consistent with prediction, knockdown of FAK induced apoptosis in the fibroblast-populated collagen matrix.     

Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro

Summary Human gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues.     

A heterocyclic molecule kartogenin induces collagen synthesis of human dermal fibroblasts by activating the smad4/smad5 pathway

Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However, only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So, we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts.     

Repair of bleomycin-damaged DNA by human fibroblasts

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.     

Computational analysis of bovine alpha-1 collagen sequences

Bovine collagen alpha-1 is a naturally occurring extracellular matrix protein found in tendons and other connective tissues. It playsa vital role in cell growth, differentiation, attachment, and migration. Recent findings have established that collagen alpha-1 isinvolved in osteogenesis imperfecta phenotype in cattle but deep information about other members of this large family is notavailable so far. So with a view to finding a new edge and attempt to figure out a correlation among the well attributed Bovinealpha-1 collagen sequences are executed and analyzed. To do so, comparative analysis among the 28 members of collagen familyhas been carried out using Computational tools. Consequently, based on the physico-chemical, secondary structural, functionaland phylogenetic classifications, we have selected collagen 12, 14 and 20 as targets for pathological conditions. These proteinsbelong to the FACIT family and significantly showed low glycine and proline content, high instability and aliphatic index.Moreover, FACIT family collagens contain multiple triple helical domains and being members of the FACIT family, bovinecollagen 12, 14, 20 do not form fibrils by themselves but they are associated to collagen 1 associated fibrils. These collagenmolecules might be crucial candidates to detect and understand the process of matrix remodeling in diseases especially in the arenaof cellular compartments.     

Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon

Summary The orderly arrangement of fibroblasts and collagen in tendons and ligaments suggests that these cells may have precise relationships with one another and with the collagen fibrils. The spatial organization of rat tail tendon was therefore examined using scanning and transmission electron microscopy and by reconstructing a 35-m long segment of tendon from serial transmission electron micrographs. Fibroblasts were regularly arranged in columns and showed more intimate association in the longitudinal than in the transverse plane. Thin cytoplasmic sheets extended up to 3 m transversely, frequently forming junctional attachments with similar processes from adjacent cells or from the same cell. Longitudinal processes were longer, often extending for more than 20 m and forming junctional attachments with other cells in the same column. Such processes often exhibited invaginations in which there were single fibrils or small groups of fibrils; this arrangement may be indicative of fibril elongation or may serve to transmit tension between the fibroblast and the collagen fibrils. This organization has interesting implications for the growth and function of other fibrous connective tissue, such as the periodontal ligament.     

Control of cell proliferation via elevated NEDD8 conjugation in oral squamous cell carcinoma

Adenosine (ADO) is an intermediary metabolite of adenosine trisphosphate degradation and a vasoactive mediator. We showed previously that ADO induces contraction and proliferation in rat mesangial cells by a mechanism involving A1 and A2 receptors. The studies concerning the effect of ADO on extracellular matrix (ECM) accumulation in mesangial cells are scarce. The purpose of our study was to evaluate the effect of ADO and the effect of the selective stimulation of A1 and A2 ADO receptors on the expression of ECM components fibronectin and collagen type I, in human and rat renal mesangial cells. Cultured human and rat renal mesangial cells were subjected to selective stimulation of A1 and A2 ADO receptors for 24 and 48 h. Fibronectin and collagen type I expression was evaluated by Western blot; total collagen synthesis was measured by [³H]-proline incorporation into collagen proteins. ADO, A1 and A2 receptor stimulation induce increases in fibronectin expression in rat mesangial cells, and A1 receptor stimulation partially inhibits fibronectin expression in serum-stimulated rat mesangial cells, without any effect in human mesangial cells. A2 receptor stimulation reduces collagen type I expression in serum-stimulated mesangial cells. Neither ADO nor A1 or A2 receptor stimulation induce significant changes in total collagen synthesis. These data suggest that ADO is not a major regulator of ECM synthesis in rat and human mesangial cells.     

Single amino acid substitutions in the C-terminus of collagen II alter its affinity for collagen IX

The structural integrity of cartilage depends on the presence of extracellular matrices (ECM) formed by heterotypic fibrils composed of collagen II, collagen IX, and collagen XI. The formation of these fibrils depends on the site-specific binding between relatively small regions of interacting collagen molecules. Single amino acid substitutions in collagen II change the physicochemical and structural characteristics of those sites, thereby leading to an alteration of intermolecular collagen II/collagen IX interaction. Employing a biosensor to study interactions between R75C, R789C or G853E collagen II mutants and collagen IX, we demonstrated significant changes in the binding affinities. Moreover, analyses of computer models representing mutation sites defined exact changes in physicochemical characteristics of collagen II mutants. Our study shows that changes in collagen II/collagen IX affinity could represent one of the steps in a cascade of changes occurring in the ECM of cartilage as a result of single amino acid substitutions in collagen II.     

Expression of phosphophoryn is sufficient for the induction of matrix mineralization by mammalian cells

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.     

Synergistic Activation of DNA Synthesis in Astrocytes by Fibroblast Growth Factors and Extracellular ATP

Abstract: The effects of extracellular ATP and polypeptide growth factors on DNA synthesis in primary cultures of rat astrocytes have been examined. It was found that ATP acts synergistically with either acidic or basic fibroblast growth factor to stimulate DNA synthesis. The specificity of this effect was demonstrated by the inability of ATP to potentiate DNA synthesis induced by platelet-derived growth factor or epidermal growth factor. ATP appears to act via P₂ purinergic receptors, because (a) it was more effective than adenosine and (b) the synergistic effect was observed with the hydrolysis-resistant P₂ agonists, ADPβS and ATPγS. The evidence suggests that extracellular ATP may be an important factor in regulating the extent of gliosis and, as such, may be involved in mechanisms of neural injury and repair.