Fibronectin Content Research Articles

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM—which exhibited a significant diversity in their TN-C content—in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74–97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Preliminary research on the regeneration of injured rabbit vocal folds after the transplantation of human amniotic epithelial cells

To investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring. hAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only ("injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively. hAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased. hAEC survived in the vocal folds lamina propria, and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.

Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy

Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of Nox4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-β1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of Nox4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. Nox4 downregulation by small-interfering RNA and inhibition of Nox4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-β1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal Nox4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active Nox4-based NADPH, 3) Nox4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via Nox4-sensitive, p38MAP kinase-dependent pathways. These findings implicate Nox4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy.

FIBRONECTIN AND LAMININ CONTENT IN THE TUNICA MEDIA OF SUBCUTANEOUS SMALL RESISTANCE ARTERIES OF PATIENTS WITH ESSENTIAL HYPERTENSION: PP.11.428

Objective: We have previously demonstrated that a pronounced fibrosis, due to an increased content in type 1 collagen, may be observed both in the tunica media and in the adventitia of subcutaneous small resistance arteries of patients with essential hypertension and with primary aldosteronism (JCEM 2006, 91:2638–2642). However, it was suggested that, in the development of hypertensive microvascular remodeling, a relevant role may be played by laminin and fibronectin vascular content, at least in animal models of hypertension. Aim of this study was to evaluate the amount of fibronectin and laminin within the tunica media of subcutaneous small arteries of normotensive subjects and essential hypertensive patients. Design and Methods: We have investigated 6 normotensive control subjects and 10 essential hypertensive patients. All subjects were submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, the fibronectin and laminin content within the tunica media was evaluated by immunohistochemistry, with subsequent image analysis (% of area stained). Results: As expected, clinic blood pressure values were higher in essential hypertensive patients (153/89 ± 7/11 mm Hg) than in normotensive controls (116/81 ± 10/5 mm Hg, P < 0.01). The media to lumen ratio was significantly increased in essential hypertensive patients (0.100 ± 0.03) compared with normotensive controls (0.06 ± 0.02, p < 0.01). Fibronectin media content was significantly greater in essential hypertensive patients (7.41 ± 2.28 %), compared with normotensive controls (5.62 ± 0.40, P < 0.05). A significant correlation was observed between fibronectin media content and media to lumen ratio (r = 0.49, p < 0.05). No significant difference in laminin media content was observed between groups (3.7 ± 1.71 % in essential hypertensive patients, 5.63 ± 1.79 % in normotensive controls). Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, fibronectin, but not laminin media content is increased. Fibronectin might be therefore involved in the development of small resistance artery remodeling in humans.

Circulating fibronectin affects bone matrix, whereas osteoblast fibronectin modulates osteoblast function

The bone matrix is composed mostly of collagen, but the initial and continuous presence of fibronectin was found to be crucial for collagen matrix integrity in vitro. It has been assumed that osteoblasts produce the fibronectin required for bone matrix formation. Using transgenic mice, we conditionally deleted fibronectin in the osteoblasts and in the liver using the cre-loxP system. We also used mice with mutated fibronectin and conditionally deleted beta(1)-integrin in osteoblasts to identify the receptor involved in fibronectin effects on osteoblasts. Conditional deletion of fibronectin in the differentiating osteoblasts [using the 2.3 kb collagen-alpha1(I) promoter] failed to show a decrease in fibronectin amount in the bone matrix despite evidence of successful deletion. Using these mice we established that osteoblast-derived fibronectin solely affects osteoblast function. This effect was not mediated by integrins that bind to the RGD motif. Conditional deletion of fibronectin in the liver showed a marked decrease in fibronectin content in the matrix associated with decreased mineral-to-matrix ratio and changed biomechanical properties but had no effect on osteoblasts or osteoclasts. In conclusion, osteoblast fibronectin affects osteoblasts function. This does not seem to be mediated by the RGD motif on fibronectin. In contrast, liver-derived fibronectin affects bone matrix properties without affecting osteoblast or osteoclast function. A novel role for liver-derived circulating fibronectin thus was defined and delineated from that of locally produced fibronectin.

Role of insulin-like growth factor binding protein 7 in the process of hepatic fibrosis promoted by activated hepatic stellate cells.

Objective To investigate the regulatory effect of insulin-like growth factor binding protein 7 (IGFBP7) on the synthesis and secretion of fibronection and the inhibiting effect of anti-IGFBP7 antibody on apop-tosis of hepatic stellate cell-T6 (HSC-T6). Methods HSC-T6 cultured in vitro were divided into blank control group, IGFBP7 20 μg/L group, anti-IGFBP7 antibody 0.25 mg/L group, anti-IGFBP7 antibody 0.50 mg/L group and anti-IGFBP7 antibody 1.00 mg/L group. HSC-T6 were treated by IGFBP7 for 24 hours, then the expression of fibronectin was detected by Western blot and the content of fibronectin by ELISA. After incubating HSC-T6 with anti-IGFBP7 antibody for 14 hours, the inhibiting effect of anti-IGFBP7 antibody on HST-T6 was evaluated by MTT assay, and the effect of anti-IGFBP7 on the apoptosis of HST-T6 was detected by flow cytometry. All data were analyzed by one-way ANOVA, t test and SNK-q test. Results The content and expression of fibronection in IGFBP7 20 mg/L group were significantly higher than those in the blank control group (t = 22.06, 7.43, P < 0.05). Anti-IGFBP7 antibody had an obvious inhibiting effect on the proliferation of HSC-T6 (F=14. 70, P < 0.05), and it also enhanced the ratio of apoptosis of HSC-T6 (F = 63.79, P < 0.05). Conclusions IGFBP7 can increase the synthesis and secretion of fibronectin which is the principal component of extracellular matrix. Anti-IGFBP7 antibody can inhibit the proliferation and promote the apoptosis of HSC-T6. Key words: Insulin-like growth factor binding protein-7; Hepatic stellate cells; Fibronectin; Apoptosis

Wound healing in oral mucosa results in reduced scar formation as compared with skin: Evidence from the red Duroc pig model and humans

Scar formation is a common, unwanted result of wound healing in skin, but the mechanisms that regulate it are still largely unknown. Interestingly, wound healing in the oral mucosa proceeds faster than in skin and clinical observations have suggested that mucosal wounds rarely scar. To test this concept, we created identical experimental wounds in the oral mucosa and skin in red Duroc pigs and compared wound healing and scar development over time. We also compared the pig oral mucosal wound healing to similar experimental wounds created in human subjects. The findings showed significantly reduced scar formation at both clinical and histological level in the pig oral mucosa as compared with skin 49 days after wounding. Additionally, the skin scars contained a significantly increased number of type I procollagen immunopositive cells and an increased fibronectin content, while the oral mucosal wounds demonstrated a prolonged accumulation of tenascin-C. Furthermore, the pig oral mucosal wounds showed similar molecular composition and clinical and histological scar scores to human oral mucosal wounds. Thus, the reduced scar formation in the pig oral mucosa provides a model to study the biological processes that regulate scarless wound healing to find novel approaches to prevent scar formation in skin.

A Plasma-Based Biomatrix Mixed with Endothelial Progenitor Cells and Keratinocytes Promotes Matrix Formation, Angiogenesis, and Reepithelialization in Full-Thickness Wounds

In search of an autologous vascularized skin substitute, we treated full-thickness wounds (FTWs) with autologous platelet-rich plasma gel (APG) in which we embedded endothelial progenitor cells (EPCs) and basal cell keratinocytes (KCs). We cultivated autologous KCs in low-serum conditions and expanded autologous EPCs from venous blood. FTWs (n = 55) were created on the backs of four pigs, covered with wound chambers, and randomly assigned to the following treatments: (1) APG, (2) APG + KCs, (3) APG + EPCs, (4) APG + KCs + EPCs, and (5) saline. All wounds were biopsied to measure neovascularization (lectin Bandeiraea Simplicifolia-1 (BS-1), alpha smooth muscle actin [alphaSMA], and membrane type 1 matrix metalloproteinase (MT1-MMP)), matrix deposition (fibronectin, collagen type I/III, and alphavbeta3), and reepithelialization. Wound fluids were analyzed for protein expression. All APG-treated wounds showed more vascular structures (p < 0.001), and the addition of EPCs further improved neovascularization, as confirmed by higher lectin, alphaSMA, and MT1-MMP. APG groups had higher collagen I/III (p < 0.05), alphavbeta3, and fibronectin content (p < 0.001), and they exhibited higher concentrations of platelet-derived growth factor subunit bb, basic fibroblast growth factor, hepatocyte growth factor, insulin growth factor-1, transforming growth factor-beta1 and -beta3, matrix metalloproteinase-1 and -z9, and tissue-inhibiting matrix metalloproteinase-1 and -2. Applying APG + KCs resulted in the highest reepithelialization rates (p < 0.001). No differences were found for wound contraction by planimetry. In this porcine FTW model, APG acts as a supportive biomatrix that, along with the embedded cells, improves extracellular matrix organization, promotes angiogenesis, and accelerates reepithelialization.

Content of Fibronectin and Glycosaminoglycans in Blood Serum of Rats with SiO2-Induced Inflammation after Radon-Containing Water Bath

In animals with SiO(2)-induced inflammation, exposure in radon-containing water promoted normalization of fibronectin content on days 3 and 10 and reduced the content of glycosaminoglycans on days 3 and 20 compared to the corresponding values in intact animals and rats with SiO(2)-induced inflammation not exposed to radon.

A Catalytic Role of Heparin within the Extracellular Matrix

We investigated the mechanism by which heparin enhances the binding of vascular endothelial growth factor (VEGF) to the extracellular matrix protein fibronectin. In contrast to other systems, where heparin acts as a protein scaffold, we found that heparin functions catalytically to modulate VEGF binding site availability on fibronectin. By measuring the binding of VEGF and heparin to surface-immobilized fibronectin, we show that substoichiometric amounts of heparin exposed cryptic VEGF binding sites within fibronectin that remain available after heparin removal. Measurement of association and dissociation kinetics for heparin binding to fibronectin indicated that the interaction is rapid and transient. We localized the heparin-responsive element to the C-terminal 40-kDa Hep2 domain of fibronectin. A mathematical model of this catalytic process was constructed that supports a mechanism whereby the heparin-induced conformational change in fibronectin is accompanied by release of heparin. Experiments with endothelial extracellular matrix suggest that this process may also occur within biological matrices. These results indicate a novel mechanism whereby heparin catalyzes the conversion of fibronectin to an open conformation by transiently interacting with fibronectin and progressively hopping from molecule to molecule. Catalytic activation of the extracellular matrix might be an important mechanism for heparin to regulate function during normal and disease states.

Role of Increased Penile Expression of Transforming Growth Factor‐β1 and Activation of the Smad Signaling Pathway in Erectile Dysfunction in Streptozotocin‐Induced Diabetic Rats

It has been suggested that transforming growth factor-beta1 (TGF-beta1) plays an important role in the pathogenesis of diabetes-induced erectile dysfunction. To investigate the expression and activity of Smad transcriptional factors, the key molecules for the initiation of TGF-beta-mediated fibrosis, in the penis of streptozotocin (STZ)-induced diabetic rats. Fifty-two 8-week-old Sprague-Dawley rats were used and divided into control and diabetic groups. Diabetes was induced by an intravenous injection of STZ. Eight weeks later, erectile function was measured by electrical stimulation of the cavernous nerve (N = 12 per group). The penis was harvested and stained with Masson trichrome or antibody to TGF-beta1, phospho-Smad2 (P-Smad2), smooth muscle alpha-actin, and factor VIII (N = 12 per group). Penis specimens from a separate group of animals were used for TGF-beta1 enzyme-linked immunosorbent assay (ELISA), P-Smad2/Smad2, phospho-Smad3 (P-Smad3)/Smad3, fibronectin, collagen I, and collagen IV western blot, or hydroxyproline determination. Erectile function was significantly reduced in diabetic rats compared with that in controls. The expression of TGF-beta1, P-Smad2, and P-Smad3 protein evaluated by ELISA or western blot was higher in diabetic rats than in controls. Compared with that in control rats, P-Smad2 expression was higher mainly in smooth muscle cells and fibroblasts of diabetic rats, whereas no significant differences were noted in endothelial cells or in the dorsal nerve bundle. Cavernous smooth muscle and endothelial cell contents were lower in diabetic rats than in controls. Cavernous fibronectin, collagen IV, and hydroxyproline content was significantly higher in diabetic rats than in controls. Upregulation of TGF-beta1 and activation of the Smad signaling pathway in the penis of diabetic rats might play important roles in diabetes-induced structural changes and deterioration of erectile function.

Inhibition of Superoxide Dismutase Induces Collagen Production in Cardiac Fibroblasts

The aim of this study was to determine whether inhibition of superoxide dismutase (SOD) with diethyldithiocarbamic acid (DETC) could affect the collagen production, the mRNA and protein expression of collagen types I and III, and fibronectin in control and angiotensin II (ANG II)-treated cardiac fibroblasts. Its effect was compared with the SOD mimetics tempol and EUK-8 and with polyethyleneglycol (PEG)-SOD. Cardiac fibroblasts were cultured to confluence, incubated in serum-free Dulbecco's modified Eagle's medium for 24 h, preincubated with(out) the tested inhibitors for 1 h and further incubated with(out) ANG II (1 micromol/l) for 24 h. DETC dose-dependently inhibited the activity of CuZn-SOD in cardiac fibroblasts. Superoxide anion production was increased by DETC and decreased by tempol in control and ANG II-treated fibroblasts. DETC also reduced the intracellular generation of reactive oxygen species (ROS) (such as H2O2, hydroxyl radicals, hydroperoxides) in control and ANG II-treated fibroblasts, whereas tempol reduced the ROS production only in ANG II-treated fibroblasts. ANG II and DETC stimulated the collagen production and the collagen I and fibronectin content in fibroblasts. The SOD mimetics tempol and EUK-8 as well as PEG-SOD reduced the collagen production. ANG II and DETC stimulated the tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 levels, whereas tempol decreased the TIMP-2 content in control and ANG II-treated fibroblasts. Matrix metalloproteinase (MMP)-1 level was reduced by ANG II and DETC and increased by tempol. These data suggest a vital role of SOD and the formed ROS in the accumulation of collagen in cardiac fibroblasts.

Altered renal morphology in transgenic mice with cholecystokinin overexpression

Although cholecystokinin is a regulatory peptide with a predominant role in the brain and the gastrointestinal tract, there is an increasing evidence for its role in the kidney. The aim of this study was to reveal morphological changes in the structure of kidney of mice with cholecystokinin overexpression by means of light, transmission and scanning electron microscope, and atomic force microscopy. Using immunohistochemistry the expression of important basement membrane proteins collagen IV, laminin and fibronectin, as well the distribution of cholecystokinin-8 in the renal structures was evaluated. The altered morphology of kidneys of mice with cholecystokinin overexpression was seen by all microscopic techniques used. The renal corpuscles were relatively small with narrow capsular lumen. The basement membranes of renal tubules were thickened and the epithelial cells were damaged, which was more pronounced for distal tubules. Characteristic feature was the increased number of vesicles seen throughout the epithelial cells of proximal and especially in distal tubules reflecting to the enhanced cellular degeneration. The relative expression of laminin but not collagen IV in the glomerular basement membrane was higher than in the tubular basement membranes. The content of fibronectin, in opposite, was higher in tubular membranes. Cholecystokinin-8 was clearly expressed in the glomeruli, in Bowman's capsule, in proximal and distal tubules, and in collecting ducts. Ultrastructural studies showed irregularly thickened glomerular basement membranes to which elongated cytopodia of differently shaped podocytes were attached. As foot processes were often fused the number of filtration pores was decreased. In conclusion, cholecystokinin plays important role in renal structural formation and in functioning as different aspects of urine production in mice with cholecystokinin overexpression are affected-the uneven glomerular basement membrane thickening, structural changes in podocytes and in filtration slits affect glomerular filtration, while damaged tubular epithelial cells and changed composition of thickened tubular basement membranes affect reabsorption.

Multivectorial Abnormal Cell Migration in the NOD Mouse Thymus

We previously described a fibronectin/VLA-5-dependent impairment of NOD thymocyte migration, correlated with partial thymocyte arrest within thymic perivascular spaces. Yet, NOD thymocytes still emigrate, suggesting the involvement of other cell migration-related alterations. In this context, the aim of this work was to study the role of extracellular matrix ligands, alone or in combination with the chemokine CXCL12, in NOD thymocyte migration. Intrathymic contents of CXCL12, fibronectin, and laminin were evaluated by immunohistochemistry while the expression of corresponding receptors was ascertained by flow cytometry. Thymocyte migration was measured using Transwell chambers and transendothelial migration was evaluated in the same system, but using an endothelial cell monolayer within the chambers. NOD thymocytes express much lower VLA-5 than C57BL/6 thymocytes. This defect was particularly severe in CD4(+) thymocytes expressing Foxp3, thus in keeping with the arrest of Foxp3(+) cells within the NOD giant perivascular spaces. We observed an enhancement in CXCL12, laminin, and fibronectin deposition and colocalization in the NOD thymus. Furthermore, we detected altered expression of the CXCL12 receptor CXCR4 and the laminin receptor VLA-6, as well as enhanced migratory capacity of NOD thymocytes toward these molecules, combined or alone. Moreover, transendothelial migration of NOD thymocytes was diminished in the presence of exogenous fibronectin. Our data unravel the existence of multiple cell migration-related abnormalities in NOD thymocytes, comprising both down- and up-regulation of specific responses. Although remaining to be experimentally demonstrated, these events may have consequences on the appearance of autoimmunity in NOD mice.

Methods for investigating integrin‐matrix interactions as a function of matrix mechanics and composition

To investigate the role of mechanics in cell signaling, we have engineered polyacrylamide (PAAM) gels with precisely tunable stiffness and fibronectin (FN) content. Varying the acrylamide / bis ratio alters PAAM stiffness, as determined with tensile testing and atomic force microscopy (AFM). To promote cell attachment, FN is covalently linked throughout the gel, and can then be quantified with an ELISA. Analyzing AFM data with the Hertz and Oliver‐Pharr models, we calculated gel elastic moduli ranging from 11 – 344 kPa and 7.5 – 95 kPa. We also found that stiffer gels incorporate more FN than softer gels, and that adding FN reduces the modulus of softer gels by ~10%. Controlling for these factors allowed for precise and independent changes in gel mechanics and FN content.To study integrin‐FN interactions on PAAM gels, we adapted published methods for quantifying integrin binding. After vascular smooth muscle cells (VSMCs) were grown on gels or FN‐coated plastic, bound FN‐alpha5 integrin units were cross‐linked and assayed with an ELISA. Preliminary data suggest that integrin activity per cell is reduced with decreasing stiffness or higher cell density. Future studies using the methods presented herein will provide insight into how VSMCs recognize and respond to changes in ECM composition and stiffness, aiding the rational design of new treatment for vascular disease. Supported by NIH grants HL56200 and HL72900.

The role of stratifin in fibroblast–keratinocyte interaction

Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a "stemness condition" of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.

Chronic intranasal administration of mould spores or extracts to unsensitized mice leads to lung allergic inflammation, hyper‐reactivity and remodelling

Allergic asthma is a serious multifaceted disease characterized by eosinophil-rich airway inflammation, airway hyperreactivity and airway wall modifications known as remodelling. We previously demonstrated that the spores of two allergenic moulds, Alternaria alternata and Cladosporium herbarum, were potent inducers of immunoglobulin E (IgE) production. Moreover, mice sensitized by two intraperitoneal injections before intranasal challenge with A. alternata or C. herbarum spores developed an allergic lung inflammation and hyperreactivity. Here we report on the effect of chronic intranasal administration of C. herbarum spores or A. alternata extracts to unsensitized BALB/c mice. Our results demonstrate that this chronic treatment led to an increase of total serum IgE and the appearance of specific IgE and IgG1. Total cell number in bronchoalveolar lavage fluid from treated mice was highly increased compared to phosphate-buffered-saline-treated mice because of the accumulation of macrophages, neutrophils, lymphocytes and eosinophils. Airway hyperreactivity appeared after 3 weeks (extract) and 7 weeks (spores) and was maintained during the whole treatment. Increased interleukin-13 mRNA expression in the lungs and T helper type 2 cytokines (interleukin-4, -5, -6 and -13) and transforming growth factor-beta secretion in bronchoalveolar lavage fluid were also observed. Lung hydroxyproline and fibronectin contents indicated increased fibrosis in mice treated with mould allergen. These observations were confirmed by histological analysis demonstrating airway wall remodelling and strong mucus production. These observations show that this model, using chronic intranasal administration of relevant particulate allergens, is an interesting tool for the study of mechanisms leading to allergic pulmonary diseases and lung remodelling.

Dynamics of phase transitions in drying drops of human serum protein solutions

The dynamics of structuring of protein solutions in drying drops and their morphology versus the composition of dissolved proteins are studied. An increase in the content of immunoglobulins and fibronectin is shown to have an effect on the physical properties of adsorption layers at the fluid-air interface and thereby changes the dynamic parameters of structuring in drying drops. These changes are embodied in the shape of the acoustomechanical impedance curves of drying drops and can be estimated numerically. Thus, experimental support is provided for the previous phenomenological data indicating that medical diagnosis based on recording the dynamics of the acoustomechanical impedance in drying drops of blood serum is a feasibility.

Fibronectin Is Not the Only Important Molecule Required for Fibrinogen/VWF-Independent Platelet Aggregation: Study of Thrombosis in a New Strain of Triple Deficient Mice.

Fibrinogen (Fg) has been considered essential for platelet aggregation. We demonstrated, however, that thrombi do form in Fg-deficient mice and in mice doubly deficient for both fibrinogen and von Willebrand factor (Fg/VWF−/−). We further reported that β3 integrin and thrombin are critical for this Fg/VWF-independent platelet aggregation. In Fg−/− or Fg/VWF−/− mice, platelet fibronectin (Fn) content is increased 3–5 fold. Furthermore, thrombus growth and stability are impaired in plasma Fn conditional deficient (M×-Cre, Fnflox/flox) mice. These data are consistent with the most recent studies of Fn assembly and suggest that Fn may support platelet thrombus formation. To examine whether Fn is the alternative key molecule which mediates platelet aggregation and thrombus formation in Fg/VWF−/− mice, we developed a novel strain of triple knockout (TKO) mice by breeding Fg/VWF−/− mice with M×-Cre+/− Fnflox/flox conditional knockout mice. Cre- littermates delivered from the same parents were used as a control. Fn depletion was induced by i.p. injections of polyinonic-polycytidylic acid. We found that TKO mice are viable with dramatically decreased levels of Fn in both the plasma (<2% of control) and platelets (<20% of control) as determined by immunoblot. No significant difference was found in peripheral blood cell counts or platelet surface adhesion proteins (GPIbα, P-selectin, β3 and β1 integrins) as compared with control mice. Unexpectedly, TKO platelet aggregation induced by thrombin and thrombin receptor activation peptide (TRAP) was enhanced in both platelet rich plasma and PIPES buffer (P<0.05). This phenomenon was also observed in a parallel-plate flow chamber. Significantly more TKO aggregates formed when fluorescently labelled whole blood was perfused over a collagen surface at 500s−1 (P<0.001). We also studied thrombus formation in TKO and Cre- control mice using intravital microscopy. Our preliminary data showed no obvious decrease in thrombus formation in TKO mice and injured arterioles occluded in one of two experimental mice. Our data suggest that Fn is not the only important molecule required for platelet aggregation and thrombus formation in Fg/VWF−/− mice. Further investigation of how Fn may inhibit Fg/VWF-independent platelet aggregation and the identification of what ligand(s) support this novel aggregation pathway may unveil an alternative haemostatic pathway in the absence of Fg and VWF.

Control of Platelet Fibronectin Internalization and Cell Surface Retention by Fibrinogen: Lessons from Hypofibrinogemic Patients.

Although fibrinogen (Fg) has been considered essential for platelet aggregation, we previously demonstrated that thrombi still formed in Fg-deficient mice. In these animals, platelet fibronectin (Fn) content is increased >3 fold, suggesting that Fn may play a role in thrombosis and hemostasis. However, it is unclear whether Fg controls human platelet Fn internalization. We recently identified severely hypofibrinogenaemic patient born from a consanguineous marriage (Xu X et al. Thromb and Haemost 2006; 95: 931–5). The patient and his father had respective plasma Fg levels of 1.7mg/dL (approximately 20 times less than the normal level) and 10.5 mg/dL. We examined the platelet Fn content of the patient, his parents, and healthy donors by immuno-electron microscopy (IEM), western blot, and flow cytometry (FACS) using a rabbit anti-human Fn-specific antibody (Sigma, USA). Significant increase of patient platelet Fn content was detected by all three methods, in which mean fluorescence intensity (MFI) of the patient's platelet Fn by FACS was 3-fold higher than healthy donors. No concomitant alteration of platelet thrombospondin-1 and vitronectin was detected. These data are consistent with our observations in animal models. To examine the role of platelet Fn content in platelet aggregation, we isolated human platelets via sepharose 2B gel filtration. Cell surface Fn expression after thrombin stimulation was examined by FACS. Surprisingly, although platelet Fn was increased, cell surface Fn was not increased. Rather, surface Fn was approximately 6-fold less than that of his father and 10-fold less than healthy donors. Using ELISA, we observed that the patient's platelet Fn was mainly released into the extracellular medium after thrombin treatment since the supernantant contained 2–3 fold higher levels of Fn than that of healthy donors (472.8 ± 13 ng/mL versus 213.5 ±4.7 ng/mL). Since fibrin has been reported as a receptor of Fn on platelet surface, we tested the hypothesis that high expression of platelet surface Fn in healthy donors was due to fibrin formation on the platelet surface after thrombin treatment. Gel filtered platelets from healthy donors were treated with either thrombin or thrombin receptor activation peptide (TRAP, at 100μM). The latter is able to induce platelet Fg release but not fibrin formation. We found that fibrin was indeed expressed on the platelet surface after thrombin but not TRAP treatment, which were detected by both FACS and IEM with a monoclonal anti-fibrin specific antibody (clone T2G1, Accurate Bilchemicals, USA). Consistent with these data, Fn was only significantly detected on the surface after thrombin but not TRAP treatment (MFI = 10.9 ± 2.5 versus 1.3 ± 0.3). To the best of our knowledge, this is first report that thrombin converts platelet-released Fg to fibrin on the platelet surface. Our data also suggested that Fg not only controls platelet Fn internalization but also regulates platelet surface Fn retention by the fibrin-Fn interaction.