In Vitro Biomineralization Induced by Self-Assembled Extracellular Matrix Proteins

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

The biomineralization process in bone involves the assembly of calcium phosphate (CP) as a bio-inorganic nanocomposite in association with extracellular matrix proteins. Towards developing synthetic systems that can promote the formation of biomimetic hydroxyapatite, the formation of CP from solution onto the surfaces of a model system composed of silica nanoparticles (SNPs) of differing physiochemical characteristics was studied. In addition to varying charge, two protein mediators collagen and phosvitin were adsorbed onto the surface of SNPs to form protein-coated SNPs, and the precipitation of CP onto the SNP from solutions containing physiologically relevant concentrations of calcium and phosphate ions was studied. A comparison of zeta potential (ζ) versus pH isotherms between the protein-coated SNP and native protein reveals deviation in ζ that can be attributed to perturbation in the protein structure. Analysis of the ζ versus pH isotherms for CP–protein–silica nanocomposites and the native CP indicates that CP deposition on SNPs occurs in a heterogeneous manner with segregated regions of CP. Interestingly, SNP composites containing phosvitin exhibit lower component segregation relative to composites containing collagen, and that the difference in segregation originates from the difference in ζ of each adsorbed protein. This suggests that protein-mediated biomineralization might be governed by surface energetics as much as biology.

Combined antiretroviral therapy (cART) attenuates hepatic fibrosis in hepatitis C virus and HIV coinfected patients. However, the role of HIV or cART on hepatic fibrosis in HIV monoinfection is discussed controversially. During liver fibrosis, matrix metalloproteinases (MMPs) degrade extracellular matrix (ECM) proteins into small soluble fragments, which reflect hepatic remodeling processes. This study used these novel biomarkers to investigate the effect of HIV and cART on hepatic fibrosis remodeling. In 249 patients with HIV monoinfection and 55 healthy controls, the serum levels of MMP-degraded collagen type III (C3M), biglycan (BGM), elastin (ELM), as well as the formation marker 7S (P4NP 7S), and MMP-degraded collagen type IV (C4M) were determined using specific ELISAs. Sixty-eight patients underwent a follow-up visit 3 years later including assessment of ECM markers and fibrosis using transient elastography (Fibroscan). C3M, BGM, C4M and P4NP 7S were significantly elevated in HIV patients compared to controls and correlated to HIV viral loads and inversely to cART duration. C4M, P4NP 7S and ELM were lower in patients under cART therapy and in patients without HIV viremia, indicating that lowering of the HIV load by cART attenuates remodeling of ECM. The levels of C3M, C4M, P4NP 7S and ELM correlated significantly with the progression of fibrosis in these patients. Specific therapy of patients with HIV monoinfection also beneficially influences liver fibrosis. These novel markers of liver fibrosis remodeling may help to monitor the hepatic effects by HIV therapy.

Extracellular matrix (ECM) proteins play an essential role in various biological processes in multicellular organisms, and their abnormal regulation can lead to many diseases. For large-scale ECM protein identification, especially through proteomic-based techniques, a theoretical reference database of ECM proteins is required. In this study, based on the experimentally verified ECM datasets and by the integration of protein domain features and a machine learning model, we developed ECMPride, a flexible and scalable tool for predicting ECM proteins. ECMPride achieved excellent performance in predicting ECM proteins, with appropriate balanced accuracy and sensitivity, and the performance of ECMPride was shown to be superior to the previously developed tool. A new theoretical dataset of human ECM components was also established by applying ECMPride to all human entries in the SwissProt database, containing a significant number of putative ECM proteins as well as the abundant biological annotations. This dataset might serve as a valuable reference resource for ECM protein identification.

RHO-ASSOCIATED KINASE (ROCK) REGULATES ENDOTHELIAL NITRIC OXIDE SYNTHASE IN BLOOD OUTGROWTH ENDOTHELIAL CELLS

The purpose of this study was to develop an in vitro model of odontoblast differentiation and dentin formation which allows to deeply and better study the process of odontoblast differentiation and protein matrix deposition and mineralization. Human dental pulp cells were obtained from extracted molar teeth of donors under informed consent. Cells were induced to odontoblast differentiation for 7, 14, 21 and 28 days by adding in the cell medium dexamethasone, β-glicerophosphate, ascorbic acid and TGF-β1. Alizarin red staining, electron microscopy, western blotting were carried out to demonstrate the odontoblast differentiation process, extracellular matrix deposition and mineralization. Alizarin red staining results showed a gradual calcium deposition during the treatment, reaching the highest signal at 28 days of stimulation. Electron microscopy demonstrated the deposition of fibrillar structures in the extracellular matrix, connected with extracellular matrix proteins. Western blotting analysis showed the expression of collagen type I and dentin matrix protein 1, markers for the odontoblast phenotype. The in vitro odontoblast differentiation model described if an highly repetitive model and it represents a potential and promising tool to explore the extracellular matrix deposition and mineralization process in physiological and pathological conditions.

The in vitro behavior of as-prepared and pre-immersed RF-sputtered calcium phosphate thin films in a rat bone marrow cell model

Dentinogenesis involves the initial odontoblastic synthesis of a collagen-rich extracellular matrix (ECM) and predentin that is converted to dentin when the collagen fibrils become mineralized. Since the width of predentin is rather uniform, we postulate that extracellular events regulate dentinogenesis. Similarly, osteogenesis involves an initial unmineralized osteoid that is mineralized and converted to bone. To gain insights into these two processes, we compared ECM proteins in bone with those in dentin, focusing upon the sialic acid (SA)-rich proteins. We observed qualitative similarities between the SA-rich proteins, but distinct differences in the amounts of osteopontin (OPN) and dentin sialoprotein (DSP). OPN, a predominant protein in bone, was found in much smaller amounts in dentin. Conversely, DSP was abundant in dentin ECM, but found sparingly in bone. Molecular cloning experiments indicate that coding sequences for DSP and dentin phosphoprotein (DPP) are found on the same mRNA. We believe that the initial form of the precursor protein DSPP is inactive in influencing the mineralization process and that it must be activated by cleavage of peptide bonds in conserved regions. Thus, unknown proteinases would act on DSPP, possibly at the mineralization front, and liberate active DPP, which plays an initiation and regulatory role in the formation of apatite crystals. This post-translational processing reaction would represent an important control point in dentinogenesis. Recently, we identified uncleaved DSPP in dentin extracts, which should allow us to test portions of our hypothesis.

We have identified a novel cellular action of thrombin on cultured rat adrenal medullary endothelial cells (RAMEC). Five-minute incubation of RAMEC with physiological concentrations of thrombin (<1 U/ml) caused within 3 h an increase in the basolateral deposition of the extracellular matrix (ECM) proteins fibronectin, laminin, and collagens IV and I, concomitant with a corresponding decrease in the apical release of these proteins into the medium. This shift in vectorial secretion of ECM proteins, quantitated with enzyme-linked immunoassays, was time dependent. Maximal stimulation of ECM protein deposition was observed after incubation of cells with thrombin for 5-15 min. Prolonged exposure (>1 h) to thrombin resulted in loss of proteins from the ECM. Thrombin-stimulated ECM protein deposition exhibited a bell-shaped dose dependence, peaking for all proteins at 0.25 U/ml of thrombin, and was independent of de novo mRNA or protein synthesis. Maximal amounts of deposited proteins increased between 2.5-fold (fibronectin) and 4-fold (collagen I) over baseline values. Similar results were obtained with thrombin receptor agonist peptide (TRAP), proteolytically active gamma-thrombin, and, to a lesser extent, other serine proteases such as trypsin and plasmin. A scrambled TRAP, proteolytically inactive PPACK-thrombin, DIP-thrombin, and type IV collagenase were ineffective. Together, these results suggest that the thrombin effects are mediated by proteolytic activation of the thrombin receptor. Possible involvement of the phospholipase C-signaling pathway in thrombin-mediated ECM protein deposition was also investigated. Inhibition or downregulation of protein kinase C (PKC) and chelation of intracellular or extracellular Ca2+ did not suppress, but rather enhanced, basal and thrombin-stimulated ECM protein deposition. Quantitative differences in augmentation of basolateral deposition by these treatments suggest differential regulatory pathways for individual ECM proteins. Our data indicate that, in cultured RAMEC, short-term activation of the thrombin receptor causes an increase in amounts of deposited ECM protein by a cellular signaling pathway that is independent of PKC activation and/or elevation of intracellular Ca2+.

Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

Low levels of expression of extracellular matrix (ECM) proteins in chorioamniotic membranes is a characteristic of prematurely ruptured membranes, a condition associated with 40% of preterm deliveries. In light of the rise in levels of glucocorticoids (GC) in amniotic fluid associated with preterm labor, in the present study we examined the effects of GC on the expression of major ECM proteins in cultures of amnion epithelial cells recovered after digestion of human term amnions. Amnion cells were maintained with and without 10(-7) mol/L dexamethasone (DEX), and levels of the ECM protein fibronectin (FN) were determined by enzyme-linked immunosorbent assay. DEX treatment reduced FN expression in amnion epithelial cells to 15-30% of control levels and reduced FN expression in placental cells to 30-50% of control levels. Conversely, DEX treatment weakly stimulated FN expression in chorion cell cultures. DEX treatment did not affect the total level of amnion cell protein, indicating that the effects of DEX in amnion cells did not result from a general reduction in protein synthesis. Cortisol and DEX reduced FN expression in amnion cells, with half-maximal effective concentrations of approximately 60 and 8 nmol/L, respectively. In immunoprecipitation studies, DEX treatment reduced FN and collagen III synthesis to 20% of control levels, suggesting that GC may coordinately reduce the synthesis of major ECM proteins in amnion cells. Similarly, DEX treatment reduced the levels of FN messenger ribonucleic acids in amnion cells to approximately 15% of control levels. DEX treatment also promoted a marked reduction in FN expression in amnion cells cultured in serum-free medium to 10-50% of control levels. Our results indicate that GC negatively regulate ECM protein expression in amnion epithelial cells, suggesting a potential role in the genesis of altered fetal membrane ECM protein expression associated with prematurely ruptured membranes.

Simple SummaryHuman breast tissue extracellular matrix (ECM) is a microenvironment essential for the survival and biological activities of mammary epithelial cells. The ECM structural features of human breast tissues remain poorly defined. In this study, we identified the structural and mechanical properties of human normal breast and invasive ductal carcinoma tissue ECM using histological methods and atomic force microscopy. Additionally, a protein hydrogel was generated using human breast tissue ECM and defined for its microstructural features using immunofluorescence imaging and machine learning. Furthermore, we examined the three-dimensional growth of normal mammary epithelial cells or breast cancer cells cultured on the ECM protein hydrogel, where the cells exhibited biological phenotypes like those seen in native breast tissues. Our data provide novel insights into cancer cell biology, tissue microenvironment mimicry and engineering, and native tissue ECM-based biomedical and pharmaceutical applications.Tissue extracellular matrix (ECM) is a structurally and compositionally unique microenvironment within which native cells can perform their natural biological activities. Cells grown on artificial substrata differ biologically and phenotypically from those grown within their native tissue microenvironment. Studies examining human tissue ECM structures and the biology of human tissue cells in their corresponding tissue ECM are lacking. Such investigations will improve our understanding about human pathophysiological conditions for better clinical care. We report here human normal breast tissue and invasive ductal carcinoma tissue ECM structural features. For the first time, a hydrogel was successfully fabricated using whole protein extracts of human normal breast ECM. Using immunofluorescence staining of type I collagen (Col I) and machine learning of its fibrous patterns in the polymerized human breast ECM hydrogel, we have defined the microstructural characteristics of the hydrogel and compared the microstructures with those of other native ECM hydrogels. Importantly, the ECM hydrogel supported 3D growth and cell-ECM interaction of both normal and cancerous mammary epithelial cells. This work represents further advancement toward full reconstitution of the human breast tissue microenvironment, an accomplishment that will accelerate the use of human pathophysiological tissue-derived matrices for individualized biomedical research and therapeutic development.

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.

Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the development of a successfully engineered bone tissue scaffold, and to date there has not been a well-established method for the quantitative examination of bone mineralization in situ. We investigated the mechanical properties of MC3T3-E1 osteoblast-like cells in vitro using shear modulation force microscopy (SMFM). The elastic modulus of the mineralizing cells increased at time points corresponding to mineral production, whereas that of the non-mineralizing cells did not vary significantly over time.

Dexamethasone inhibits prolonged survival and autocrine granulocyte-macrophage colony-stimulating factor production by human eosinophils cultured on laminin or tissue fibronectin