Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Colocalization of doublecortin with the microtubules: an ex vivo colocalization study of mutant doublecortin

Doublecortin (DCX) plays an important role in neuronal migration and development, and the participation of DCX in neuronal migration has been demonstrated by intensive mutational analysis for patients with X-linked or sporadic lissencephaly, and/or subcortical laminar heterotopia. Although a previous search for protein similarity showed that DCX has a region homologous to the putative Ca(2+)/calmodulin-dependent protein kinase, the function of the DCX gene (DCX) has remained unknown. We show here that mouse DCX colocalizes with the microtubules and provide evidence that its conformational structure is important for its subcellular localization by means of mutant doublecortin expression study. The results of our study may suggest that the cytoskeleton involving DCX mediates the neuronal migration during brain development.     

A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B₂, 6-keto-Prostaglandin F_1α, and Prostaglandin E₂ release have been quantitated from cultured adult by bovine endothelial cell monolayers and from $\text{ex Vivo}$ vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF_1α released was diminished compared to amounts released from the vessel segments while thromboxane B₂ and prostaglandin E₂ release were similar in the two endothelial model systems. However, the amount of thromboxane B₂ assayed was small and the quantity of thromboxane A₂ it represents is probably of little $\text{in Vivo}$ significance to prostacyclin.     

A comparative study of thromboxane and prostacyclin release from ex vivo and cultured bovine vascular endothelium

Thromboxane B2, 6-keto-Prostaglandin F1 alpha, and Prostaglandin E2 release have been quantitated from cultured adult bovine endothelial cell monolayers and from ex Vivo vascular segments employing specific radioimmunoassays and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF1 alpha released was diminished compared to amounts released from the vessel segments while thromboxane B2 and prostaglandin E2 release were similar in the two endothelial model systems. However, the amount of thromboxane B2 assayed was small and the quantity of thromboxane A2 it represents is probably of little in vivo significance compared to prostacyclin.     

Cyclic stretch downregulates arterial vascular connexin43 protein expression: an ex vivo study

Vascular cells may communicate through gap junctions that are formed by connexin (Cx) proteins. We investigated differential regulation of arterial gap junctions by steady and cyclic stretch and the underlying mechanotransduction pathways. Ex vivo culture of rabbit thoracic aortas was used to investigate regulation of Cx43 by cyclic stretch. After culturing for 6 or 24 h, Cx43 protein levels were quantified using Western blot. Cultures under a pulsatile pressure (mean 80 mmHg, pulse 30 mmHg) decreased Cx43 protein at both 6 and 24 h as compared with cultures under a steady pressure (80 mmHg). The regulation of Cx43 protein was mediated by pulsatile pressure-induced cyclic stretch, not by cyclic stress. Protein levels of active and total Src were also decreased by cyclic stretch at 24 h. The Src- specific inhibitor PP1 in steady culture only or in both steady and pulsatile culture conditions eliminated the difference in Cx43 protein levels between the two culture conditions. Addition of reactive oxygen species inhibitor apocynin to the pulsatile culture abolished the differences in Src and Cx43 protein levels between the two cultures. Thus, Src and reactive oxygen species appear to play a role in cyclic stretch-mediated regulation of Cx43 protein. These results are likely to have important implications in cardiovascular physiology and pathophysiology under conditions wherein significant alterations in the level of cyclic stretch are present.     

Hip joint centre location: An ex vivo study

The human hip joint is normally represented as a spherical hinge and its centre of rotation is used to construct femoral anatomical axes and to calculate hip joint moments. The estimate of the hip joint centre (HJC) position using a functional approach is affected by stereophotogrammetric errors and soft tissue artefacts. The aims of this study were (1) to assess the accuracy with which the HJC position can be located using stereophotogrammetry and (2) to investigate the effects of hip motion amplitude on this accuracy. Experiments were conducted on four adult cadavers. Cortical pins, each equipped with a marker cluster, were implanted in the pelvis and femur, and eight skin markers were attached to the thigh. Recordings were made while an operator rotated the hip joint exploiting the widest possible range of motion. For HJC determination, a proximal and a distal thigh skin marker cluster and two recent analytical methods, the quartic sphere fit (QFS) method and the symmetrical centre of rotation estimation (SCoRE) method, were used. Results showed that, when only stereophotogrammetric errors were taken into account, the analytical methods performed equally well. In presence of soft tissue artefacts, HJC errors highly varied among subjects, methods, and skin marker clusters (between 1.4 and 38.5 mm). As expected, larger errors were found in the subject with larger soft tissue artefacts. The QFS method and the distal cluster performed generally better and showed a mean HJC location accuracy better than 10 mm over all subjects. The analysis on the effect of hip movement amplitude revealed that a reduction of the amplitude does not improve the HJC location accuracy despite a decrease of the artefact amplitude.     

A comparative study of ex vivo skin optical clearing using two‐photon microscopy

Multiphoton tomography (MPT) is a prospective tool for imaging the skin structure. Aiming to increase the probing depth, a comparative ex vivo study of optical clearing of porcine ear skin was performed by using two optical clearing agents (OCAs), i.e., glycerol and iohexol (Omnipaque^TM) at different concentrations, which exhibit different osmotic properties. The results show that a topical application of glycerol or Omnipaque^TM solutions onto the skin for 60 min significantly improved the depth and contrast of the MPT signals. By utilizing 40%, 60% and 100% glycerol, and 60% and 100% Omnipaque^TM it was demonstrated that both agents improve autofluorescence and SHG (second harmonic generation) signals from the skin. At the applied concentrations and agent time exposure, glycerol is more effective than Omnipaque^TM. However, tissue shrinkage and cell morphology changes were found for highly concentrated glycerol solutions. Omnipaque^TM, on the contrary, increases the safety and has no or minimal tissue shrinkage during the optical clearing process. Moreover Omnipaque^TM allows for robust multimodal optical/X‐ray imaging with automatically matched optically cleared and X‐ray contrasted tissue volumes. These findings make Omnipaque^TM more prospective than glycerol for some particular application.

     

Differential expression of extracellular matrix proteins in senescent and young human fibroblasts: A comparative proteomics and microarray study

The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.     

Immunohistochemical identification of an extracellular matrix scaffold that microguides capillary sprouting in vivo.

To gain insight into how a naturally occurring scaffold composed of extracellular matrix (ECM) proteins provides directional guidance for capillary sprouting, we examined angiogenesis in whole-mount specimens of rat mesentery. Angiogenesis was studied in response to normal maturation, the injection of a mast cell degranulating substance (compound 48/80), and mild wounding. Confocal microscopy of specimens immunolabeled for elastin revealed a network of crosslinked elastic fibers with a density of 140.8 +/- 37 mm of fiber/mm(2) tissue. Fiber diameters ranged from 180 to 1400 nm, with a mean value of 710 +/- 330 nm. Capillary sprouts contained CD31- and OX-43-positive endothelial cells as well as desmin-positive pericytes. During normal maturation, leading endothelial cells and pericytes were in contact and aligned with an elastic fiber in approximately 80-90% of all sprouts. In wounding and compound 48/80-treated specimens, in which angiogenesis was markedly increased, leading endothelial cells remained in contact and aligned with elastic fibers in approximately 60-80% of all sprouts. These observations indicate that elastic fibers are used for endothelial and pericyte migration during capillary sprouting in rat mesentery. The composition of this elastic fiber matrix may provide important clues for the development of tissue-engineered scaffolds that support and directionally guide angiogenesis.     

A phenomenological mixture model for biosynthesis and linking of cartilage extracellular matrix in scaffolds seeded with chondrocytes

A phenomenological mixture model is presented for interactions between biosynthesis of extracellular matrix (ECM) constituents and ECM linking in a scaffold seeded with chondrocytes. A system of three ordinary differential equations for average apparent densities of unlinked ECM, linked ECM and scaffold is developed along with associated initial conditions for scaffold material properties. Equations for unlinked ECM synthesis and ECM linking include an inhibitory mechanism where associated rates decrease as unlinked ECM concentration in the interstitial fluid increases. Linking rates are proposed to depend on average porosity in the evolving tissue construct. The resulting initial value problem contains nine independent parameters that account for scaffold biomaterial properties and interacting mechanisms in the engineered system. Effects of parameter variations on model variables are analyzed relative to a baseline case with emphasis on the evolution of solid phase apparent density, which is often correlated with the compressive elastic modulus of the tissue construct. The new model provides an additional quantitative framework for assessing and optimizing the design of engineered cell-scaffold systems and guiding strategies for articular cartilage tissue engineering.     

The extracellular matrix: an underexplored but important proteome

The following article is a correction to a previously published version: Bernardini G, Braconi D, Lusini P, Santucci A. Postgenomics of Neisseria meningitidis: an update. Expert Rev. Proteomics 6(2), 135–143 (2009). These corrections were made owing to concerns being raised regarding similarity between sections of the text with previously published works.

For clarity, the corrected article is published in full below. The sections in bold text correspond to the corrected sections and are therefore different to the previously published version.

Neisseria meningitidis infection still remains a major life-threatening bacterial disease worldwide. The availability of bacterial genomic sequences generated a paradigm shift in microbiological and vaccines sciences, and post-genomics (comparative genomics, functional genomics, proteomics and a combination/evolution of these techniques) played important roles in elucidating bacterial biological complexity and pathogenic traits, at the same time accelerating the development of therapeutic drugs and vaccines. This article summarizes the most recent technological and scientific advances in meningococcal biology and pathogenesis aimed at the development and characterization of vaccines against the pathogenic meningococci.     

Dehydration-responsive reversible and irreversible changes in the extracellular matrix: comparative proteomics of chickpea genotypes with contrasting tolerance

Dehydration is the most crucial environmental factor that limits plant growth, development, and productivity affecting agriculture throughout the world. Studies on genetic variations for dehydration tolerance in plants is crucial because divergent cultivars with contrasting traits aid the identification of key cellular components that confer better adaptability. The extracellular matrix (ECM) is a dynamic structure that serves as the repository for important signaling components and acts as a front-line defense. To better understand dehydration adaptation, a proteomic study was performed on the extracellular matrix of ICCV-2, a dehydration-susceptible genotype of chickpea. The proteome was generated with ECM-enriched fractions using two-dimensional gel electrophoresis. The LC-ESI-MS/MS analysis led to the identification of 81 dehydration-responsive proteins. The proteome was then compared with that of JG-62, a tolerant genotype. Comparative proteomics revealed genotype-specific expression of many proteins involved in a variety of cellular functions. Further, the reversible and irreversible changes in the proteomes revealed their differing ability to recover from dehydration-induced damage. We propose that cell wall restructuring and superior homeostasis, particularly the management of reactive oxygen species, may render better dehydration-adaptation. To our knowledge, this is the first report on the comprehensive comparison of dehydration-responsive organellar proteome of two genotypes with contrasting tolerance.     

Undulin, an extracellular matrix glycoprotein associated with collagen fibrils

Undulin, a novel noncollagenous extracellular matrix protein, was isolated from skin and placenta. In polyacrylamide gels most of the unreduced protein migrates with Mr above 1,000,000 yielding bands A (Mr 270,000), B1 (Mr 190,000), and B2 (Mr 180,000) after reduction. Undulin is biochemically and immunochemically distinct from other previously characterized large matrix glycoproteins. Immunoblotting using monoclonal antibodies suggests that bands A and B are closely related. Electron microscopy reveals undulin as structures consisting of an approximately 80-nm-long-tail with a nodule on one end and with one or two shorter arms on the other. Ultrastructurally immunolabeled undulin is found mainly between densely packed mature collagen fibrils. Indirect immunofluorescence shows bundles of uniform wavy fibers in dense connective tissues superimposable on a subpopulation of type I collagen structures. This suggests that undulin serves a specific yet unknown function in the supramolecular organization of collagen fibrils in soft tissues.     

Effects of systemic glucocorticosteroids on peripheral neutrophil functions in asthmatic subjects: an ex vivo study

In 21 asthmatic subjects, several functions of isolated peripheral neutrophils (chemokinesis and chemotaxis toward 10% E. coli; superoxide anion generation after PMA; leukotriene B(4) (LTB(4)) release from whole blood and isolated neutrophtls, before and after different stimuli) were evaluated during an acute exacerbation of asthma, and after 14 - 54 days of treatment with systemic glucocorticosteroids (GCS). During acute exacerbation, superoxide anion generation was higher in asthmatics than in eleven normal subjects (39.2 +/- 14.1 vs. 25.2 +/- 7.3 nmol, p < 0.05); there was a significant correlation between FEV(1) (% of predicted) and neutrophil chemotaxis (r = -0.52, p = 0.04). After treatment, there was no significant change in all neutrophil functions, except for a decrease in neutrophil chemotaxis in subjects who showed an FEV(1) increase > 20% after GCS treatment (from 131 +/- 18 to 117 +/- 21 mum, p = 0.005). Chemokinesis sicantly decreased in all subjects, and the changes significantly correlated with an arbitrary score of the total administered dose of GCS (r = 0.57, p < 0.05). These data suggest that neutrophil activation plays a minor role in asthma, and that treatment with GCS is not able to modify most functions of peripheral neutrophils in asthmatic subjects; chemotaxis seems to be related only to the severity of the asthma and it could reflect the improvement of the disease.     

Microstructural and mechanical insight into atherosclerotic plaques: an ex vivo DTI study to better assess plaque vulnerability

Non-invasive microstructural characterisation has the potential to determine the stability, or lack thereof, of atherosclerotic plaques and ultimately aid in better assessing plaques’ risk to rupture. If linked with mechanical characterisation using a clinically relevant imaging technique, mechanically sensitive rupture risk indicators could be possible. This study aims to provide this link–between a clinically relevant imaging technique and mechanical characterisation within human atherosclerotic plaques. Ex vivo diffusion tensor imaging, mechanical testing, and histological analysis were carried out on human carotid atherosclerotic plaques. DTI-derived tractography was found to yield significant mechanical insight into the mechanical properties of more stable and more vulnerable microstructures. Coupled with insights from digital image correlation and histology, specific failure characteristics of different microstructural arrangements furthered this finding. More circumferentially uniform microstructures failed at higher stresses and strains when compared to samples which had multiple microstructures, like those seen in a plaque cap. The novel findings in this study motivate diagnostic measures which use non-invasive characterisation of the underlying microstructure of plaques to determine their vulnerability to rupture.

Graphic abstract

     

Compromising human skin in vivo and ex vivo to study skin barrier repair

Ex vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics the lipid properties (e.g. lipid composition and lipid ordering) of the in vivo situation are not known. Here, we examined using a comprehensive ceramide analysis whether human ex vivo regenerated SC showed similar lipid properties as human in vivo regenerated SC. Both in vivo and ex vivo regenerated SC had an altered ceramide subclass composition, with increased percentages of sphingosine-based subclass and decreased percentages of phytosphingosine-based subclass ceramides, a reduced mean ceramide chain length, and a higher percentage of unsaturated ceramides. Overall, regenerated SC ex vivo showed more pronounced but similar changes compared to the in vivo response. One of the purposes of these models is to use them to mimic compromised skin of inflammatory skin diseases. The altered lipid properties in regenerated SC were comparable to those observed in several inflammatory skin diseases, which makes them a valuable model for the barrier properties in inflammatory skin diseases.     

Characterization of tissue response and in vivo degradation of cholecyst-derived extracellular matrix

A rat subcutaneous implantation model was used to evaluate the in vivo degradation and tissue response of cholecyst-derived extracellular matrix (CEM). This response was compared to that of glutaraldehyde (GA) cross-linked CEM and porcine heart valve (HV), which are designated as GAxCEM and GAxHV, respectively. Tissue composition, inflammatory cell distribution, and angiogenesis at the implant site were quantified using stereological parameters, thickness (Ta), volume fraction (Vv), surface density (Sv), length density (Lv), and radius of diffusion (Rdiff). CEM was completely infiltrated with host tissue at 21 days and resorbed by 63 days. GAxCEM was also infiltrated with host tissue, while GAxHV matrix was impermeable to host tissue infiltration. Both GAxCEM and GAxHV retained their scaffold integrity until 63 days with no apparent degradation. A fibrous tissue of thickness <52 mum, rich in collagen and vasculature, surrounded all scaffolds, and from 21 to 63 days the fibrous tissue showed maturation with a significant increase in their fibrocyte content. No signs of acute inflammatory response were observed in the study period for any of the scaffolds, while the chronic inflammatory response was predominated with macrophages for all scaffolds except for CEM at 63 days. A higher degree of giant cell formation was observed with GA cross-linked scaffolds. From 21 to 63 days, lymphocytic response decreased for CEM, while it increased significantly for GAxHV. Angiogenesis/neo-vascularization was uniform for CEM (reaching the core), significantly lower for GAxCEM within the implant area as compared to CEM, while restricted to the exterior of GAxHV matrix. In summary, CEM was a fast degrading scaffold that induced a transitional inflammatory response accompanied by gradual resorption and replacement by host connective tissue as compared to the very slow degrading GA cross-linked controls, GAxCEM and GAxHV, which caused a sustained chronic inflammatory response and remained at the site of implantation until the end of the study period of 63 days.