ED-B Fibronectin Research Articles

The fibronectin ED-A domain enhances recruitment of latent TGF-β-binding protein-1 to the fibroblast matrix.

Dysregulated secretion and extracellular activation of TGF-β1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-β-binding protein-1 (LTBP-1) and thus stores TGF-β1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-β1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-β1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.

Contrast Media Research 2017 Durango, Colorado USA, October 22–25, 2017 Convener: Michael F. Tweedle, PhD

Contrast Media Research 2017 Durango, Colorado USA, October 22–25, 2017 Convener: Michael F. Tweedle, PhD

ED-B fibronectin expression is a marker of epithelial-mesenchymal transition in translational oncology.

Fibronectin is a component of the extracellular matrix that links collagen fibers to integrins on the cell's surface. The splicing isoforms, containing the ED-B domain, are not expressed in adult tissues but only in tumor stroma or during embryonic development. Fibroblasts and endothelial cells express ED-B fibronectin during angiogenesis. Also cancer cells can synthetize ED-B fibronectin, but its function in tumor growth needs to be further elucidated.We evaluated the expression of ED-B fibronectin in prostate cancer cell lines: PC3 and DU145. Using TGF-β, we induced epithelial to mesenchymal transition in culture and observed an increase of ED-B fibronectin expression. Thereafter, we evaluated the expression of ED-B fibronectin in multipotent mesangiogenic progenitor cells, and in mesenchymal stromal cells. The expression of ED-B fibronectin was much higher in mesenchymal than prostate cancer cells even after the epithelial to mesenchymal transition.Epithelial to mesenchymal transition is a key step for tumor progression contributing to the metastatic spread. Therefore, circulating cancer cells could seed into the metastatic niche taking advantage from the ED-B fibronectin that secrete their own.

Self-assembled nanoparticles covalently consisting of doxorubicin and EDB fibronectin specific peptide for solid tumour treatment

We developed a facile modality to prepare nanoparticles consisting of doxorubicin and ZD2 motif for treating solid tumours. The nanoparticles showed great preferential cellular uptake in PC3 cells, high cell suppression, and strong anti-tumour ability.

EDB Fibronectin Specific Peptide for Prostate Cancer Targeting.

Extradomain-B fibronectin (EDB-FN), one of the oncofetal fibronectin (onfFN) isoforms, is a high-molecular-weight glycoprotein that mediates cell adhesion and migration. The expression of EDB-FN is associated with a number of cancer-related biological processes such as tumorigenesis, angiogenesis, and epithelial-to-mesenchymal transition (EMT). Here, we report the development of a small peptide specific to EDB-FN for targeting prostate cancer. A cyclic nonapeptide, CTVRTSADC (ZD2), was identified using peptide phage display. A ZD2-Cy5 conjugate was synthesized to accomplish molecular imaging of prostate cancer in vitro and in vivo. ZD2-Cy5 demonstrated effective binding to up-regulated EDB-FN secreted by TGF-β-induced PC3 cancer cells following EMT. Following intravenous injections, the targeted fluorescent probe specifically bound to and delineated PC3-GFP prostate tumors in nude mice bearing the tumor xenografts. ZD2-Cy5 also showed stronger binding to human prostate tumor specimens with a higher Gleason score (GS9) compared to those with a lower score (GS 7), with no binding in benign prostatic hyperplasia (BPH). Thus, the ZD2 peptide is a promising strategy for molecular imaging and targeted therapy of prostate cancer.

Secretory expression of a bispecific antibody targeting tumor necrosis factor and ED-B fibronectin in Pichia pastoris and its functional analysis.

Specific targeting of tumor necrosis factor (TNF)-α antagonist to the inflamed site could increase its efficacy and reduce side-effects. Here, we constructed a bispecific diabody (BsDb) that targets TNF-α and ED-B-containing fibronectin, a fibronectin isoform specifically expressed in the pannus of the inflamed synovium in rheumatoid arthritis. BsDb was secreted from Pichia pastoris as functional protein and was purified to homogeneity. BsDb could simultaneously bind to human TNF-α and B-FN and neutralize TNF-α action. Additionally, BsDb showed a significant gain both in the antigen-binding affinity and in TNF-α-neutralizing ability as compared to its original antibodies, L19 and anti-TNF-α scFv, which were produced in E. coli. BsDb was constructed and was endowed with enhanced bioactivities and improved production processing. Therefore, it holds great potential for in vivo applications.

A novel bispecific antibody targeting tumor necrosis factor α and ED-B fibronectin effectively inhibits the progression of established collagen-induce arthritis

Specific delivery of TNF-α antagonist to the inflamed site can increase its efficacy and reduce the side effects. In this study, we constructed a bispecific diabody (BsDb) that targets TNF-α and ED-B-containing fibronectin (B-FN), a fibronectin isoform specifically expressed in the pannus of the inflamed joint in rheumatoid arthritis. BsDb was produced in Escherichia coli as inclusion bodies, purified to homogeneity, and refolded to the functional form. Our data demonstrate that BsDb could simultaneously bind to human TNF-α and B-FN and neutralize TNF-α action. In the collagen-induced arthritis mouse model, we compared the biodistrubtion and therapeutic efficacy of BsDb with those of the anti-TNF-α scFv (TNF-scFv). Similar to TNF-scFv, BsDb penetrated into the synovial tissue quickly, showing a rapid clearance from blood and normal organs. In contrast, BsDb could selectively accumulate and retain in arthritic joints of mice for a long period time, resulting in a much stronger inhibition of arthritis progression in mice than TNF-scFv. The findings described herein indicate that BsDb has a good specificity to the inflamed joint, with low toxicity to normal organs and seems to be an ideal biological agent for the treatment of RA and other chronic inflammatory disease.

Regulation of Vascular Endothelial Growth Factor Expression by Extra Domain B Segment of Fibronectin in Endothelial Cells

Diabetic retinopathy entails proliferation of vascular endothelial cells (ECs) and unregulated angiogenesis. We have previously shown that ECs increase the expression of an embryonic variant of fibronectin (FN), called extra domain-B FN (ED-B FN) in response to high glucose. We also showed that ED-B FN regulates EC tube morphogenesis, possibly through vascular endothelial growth factor (VEGF). In the present study, we have attempted to decipher the mechanisms by which ED-B FN may modulate EC phenotype. We hypothesized that ED-B FN regulates VEGF expression in ECs through interaction with selected integrin receptors. To test this hypothesis, we first cultured ECs in high levels of glucose to investigate for any alteration. We then used integrin-specific matrix mimetic peptides, neutralizing antibodies, and RNAi to identify the integrin(s) involved in VEGF expression. Finally, we used an animal model of diabetes to study whether these in vitro mechanisms also take place in the retina. Our results show that exposure of ECs to high levels of glucose increased VEGF expression. ED-B FN mediated this increase since knockdown of ED-B FN completely prevented glucose-induced VEGF expression. We then identified β1 integrin as the essential receptor involved in high glucose-induced VEGF expression. We also showed that diabetes increased β1 integrin and VEGF expression in the retina, which normalized upon ED-B knockdown. These findings showed that high levels of glucose in diabetes increased VEGF expression in ECs through ED-B FN and β1 integrin interaction. These results provide novel mechanistic basis of increased VEGF expression in diabetes.

Abstract 5544: ED-B Fibronectin Spect Imaging Detects Aortic Plaque Formation in vivo

The extracellular matrix protein ED-B fibronectin (ED-B) is upregulated in inflammatory atherosclerotic lesions and is associated with local macrophage accumulation. In the present study we evaluated whether a conjugate of 99mTc with the single chain antibody against ED-B (99mTC-anti-ED-B) can be used for in vivo detection of atherosclerotic plaque lesions in apolipoprotein E- deficient mice (apoE −/− ) compared to wildtype control mice (apoE +/+ ). Methods: 12 month old apoE −/− and apoE +/+ mice were studied by SPECT 4 hours after injection of 148 MBq of 99mTC-anti-ED-B. Finally mice were sacrificed and analyzed by autoradiography, histology, immunohistochemistry (IHC) and morphometry. Results: In vivo SPECT imaging of apoE −/− mice demonstrated a significant signal activity in the thoracic area which colocalizes with the aortic arch and the supraaortic arteries. The activity corresponded to aortic lesion formation was 52.2±40.6 (1/1*ccm). Low activity was observed in apoE +/+ 9.4±4.9 (1/1*ccm), which had no significant lesion formation. The strongest signals were detected in the aortic root, the aortic arch and along the abdominal part of the aorta from apoE −/− . No vascular signal activity was seen in apoE +/+ after injection of 99mTC-anti-ED-B. The autoradiographic analysis of aortas from apoE −/− and apoE +/+ , confirmed the in-vivo observation and demonstrated signal localization in typical plaque lesions, which significantly correlated with ED-B immunoreactivity, plaque lesion (r = 0.82, P<0.01) and local macrophage accumulation (IHC with Mac3 r = 0.89, P<0.01). There was a significant correlation between ED-B-radioactivity and macrophages (r= 0.93, P<0.01). Conclusion: SPECT imaging using a 99mTc labelled antibody against ED-B fibronectin is a robust method to detect and assess inflammatory plaque lesion formation in-vivo and correlates with local macrophage accumulation.

Impact of VEGF‐dependent tumour micro‐environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice

Fibronectin (FN) is an extracellular matrix cell-adhesive glycoprotein. The alternative spliced isoform EDB-FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro-environments on EDB-FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet-FGF2 cells overexpressing fibroblast growth factor-2 (FGF2) driven by the tetracycline-responsive promoter were further transfected with a VEGF antisense cDNA, generating AS-VEGF/Tet-FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast-growing, highly vascularized Tet-FGF2 tumours. Down-regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS-VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro-environment. Quantitative RT-PCR analysis using human EDB-FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up-regulation of EDB-FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki-1 cells. However, in vivo down-regulation of VEGF expression, as occurs in AS-VEGF/Tet-FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline-treated Tet-FGF2 tumour-bearing animals, causes significant inhibition of EDB-FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT-PCR analysis. Accordingly, treatment of Tet-FGF2 tumour-bearing animals with the neutralizing anti-murine VEGF receptor-2 antibody DC101, or of Caki-1 tumour-bearing animals with the anti-VEGF antibody bevacizumab, inhibited EDB-FN expression in tumour grafts. EDB-FN down-regulation was paralleled by a decrease in vascularity, thus confirming EDB-FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro-environment, and in particular the VEGF/VEGFR-2 system, plays a key role in modulating EDB-FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB-FN in combination with anti-angiogenic and/or cytotoxic drugs.

Abstract 1735: Targeted Application of Interleukin-2 Activates T-Lymphocytes and Reduces Plaque Formation

A newly developed fusion protein (L19-IL2) consisting of an anti-ED-B fibronectin single chain antibody and the cytokine interleukin-2 (IL2), is a potent antitumor agent for renal cell cancer. ED-B fibronectin (ED-B) is expressed in inflammatory plaque lesions. Preliminary studies suggested that delivering IL2 locally to atherosclerotic tissue seem to reduce plaque progression. Therefore we investigated the application of L19-Il2 on lesion formation in apoE-deficient mice (apoE −/− ). 6-month old apoE −/− , fed with high fat diet (n = 5/group) were injected intravenously L19-IL2 (4290IU/g bodyweight), with L19 (antibody alone), with the control fusion protein D1-IL2 (4290IU/g bodyweight) or NaCL (control) at day 1, 3 and 5 and were sacrificed at day 7. Plaque lesion formation was analyzed by histology, immunohisto-chemistry (Mac3 = macrophages, CD31; actin, human IL2, ED-B, CD25, CD4), morphometry of the aortic root and by Sudan stain of the thoracic aorta followed by densitometry. Macrophage, ED-B, CD4, CD25 (IL-2 receptor subunit) content was analysed after immunohistochemistry. Results: Treatment with L19-IL2, L19, and D1-IL2 did not affect bodyweight, survival of mice compared to control (NaCL). Serum levels for blood glucose, cholesterol, liver and cardiac enzymes, and troponin were not different between L19-IL2, L19, and NaCL. Cardiac histology was also unaffected. Human IL2 was only detectable in plaque lesions after L19-IL2, but not after L19 or D1-IL2 treatment. L19-IL2 application significantly reduced plaque lesion size in the aortic root (L19-Il2: 23.2 ± 1.7%, L19: 35.2 ± 1.2%, D1-IL2: 31.6 ± 3.0%, NaCL: 31.0 ± 3.2%, p < 0.01), plaque extension in the thoracic aorta (L19-Il2: 5.3 ± 0.7%, L19: 8.2 ± 0.4%, D1-IL2: 9.8 ± 1.0%, NaCL: 8.9 ± 1.2%, p < 0.01). Mac3 immunoreactivity was reduced under L19-IL2, whereas CD4 and CD25 were strongly increased compared to L19, D1-IL2 and NaCL treated groups. Conclusion: A fusion protein, which delivers human IL-2 into plaque lesions, induces T-lymphocyte activation, reduction of macrophages and a reduction in plaque growth. The present study suggests that local application of IL2 activates cellular mechanisms involving T-lymphocytes leading to a reduction of atherosclerotic disease.

PARP activation and the alteration of vasoactive factors and extracellular matrix protein in retina and kidney in diabetes

The development of diabetic complications is associated with increased oxidative stress which may damage DNA leading to the activation of nuclear enzyme poly (ADP-ribose) polymerase (PARP). PARP overactivation may further exacerbate the oxidative state of the cell through its consumption of nicotinamide adenine dinucleotide. In diabetic retinopathy and nephropathy, early characteristic features include increased production of vasoactive factors such as endothelin 1 (ET-1) and increased synthesis of extracellular matrix (ECM) proteins such as fibronectin (FN) and its splice variant extra domain B containing (EDB(+)) FN. We investigated the role of PARP in the development of diabetic retinopathy and nephropathy. Two models of diabetic complications were used. PARP-1 knockout mice and their respective wild type controls were fed a 30% galactose diet for 2 months. The rats were given injections of PARP inhibitor 3-aminobenzamide (30 mg/kg/day). Analysis of the retinal and kidney tissues showed hyperhexosemia-induced oxidative stress and increased expression of ET-1, FN and EDB(+) FN in association with increased transcriptional co-activator p300 along with p300-dependent transcription factors, myocyte enhancing factors 2A and 2C. Furthermore, we showed increased PARP expression in the kidneys and retina of the diabetic rats. PARP blockade in both animal models prevented these hyperhexosemia-induced effects. These findings suggests that hyperhexosemia and diabetes causes upregulation of ET-1, FN and EDB(+) FN at the transcriptional level in the retina and kidney via a signaling pathway mediated by PARP and an epigenetic mechanism involving p300 and MEF2 transcription factors. Understanding these mechanisms is important in identifying novel treatment targets.

Monitoring Therapeutical Intervention with Ezetimibe Using Targeted Near-Infrared Fluorescence Imaging in Experimental Atherosclerosis

Ezetimibe (EZE), an inhibitor of cholesterol absorption, reduces atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. The matrix protein ED-B fibronectin (ED-B) is upregulated in atherosclerotic lesions. Using a novel conjugate for near-infrared fluorescence (NIRF) imaging targeting ED-B, we studied the effect of EZE on plaque lesion formation in apoE(-/-) mice. ApoE(-/-) mice received EZE (5 mug/kg/d) or chow up to the age of 4, 6, and 8 months. NIRF imaging of aortic lesions was performed 24 hours after intravenous application ex vivo and in vivo. Plaque lesion formation was analyzed by histology and immunohistochemistry. Aortic lesion formation detected by Sudan staining and NIRF imaging was significantly reduced at 6 and 8 months (p < .001). Plaque areas determined by NIRF imaging significantly correlated with Sudan staining (p < .001). EZE treatment resulted in a significant reduction in plaque macrophage and ED-B immunoreactivity (both p < .05) in brachiocephalic lesions. There was a significant reduction in plaque size in brachiocephalic arteries in 8-month-old mice treated with EZE compared with mice during short-term treatment (p < .05), indicating EZE plaque regression. Targeted NIRF imaging showed a correlation to histologic lesion extension during therapeutical intervention in experimental atherosclerosis.

ED-B fibronectin (ED-B) can be targeted using a novel single chain antibody conjugate and is associated with macrophage accumulation in atherosclerotic lesions

It has been shown that ED-B fibronectin (ED-B) is a potential target for plaque imaging. The aim of this study was to test a novel modified single chain anti-ED-B antibody (scFv) conjugated for near infrared fluorescence imaging (NIRF) with tetrasulfonated carbocyanine-maleimide (TSC-scFv) and to examine the association of ED-B with the presence of macrophages in a murine model of atherosclerosis. Expression of ED-B was observed in plaque areas in apolipoprotein E-deficient (apoE(-/-)) mice which increased with age and plaque load. Robust imaging was possible after explantation of the aorta and demonstrated a strong NIRF signal intensity in focal aortic and brachiocephalic plaque lesions, whereas no signals were found in undiseased areas. Plaque lesion ED-B was expressed by smooth muscle cell and was closely associated to macrophage infiltrates. Although not expressed by the same cell type, there was a significant correlation (p<0.01) between ED-B and macrophage immunoreactivity. In vitro human coronary and mouse smooth muscle cells significantly increased ED-B expression after angiotensin II and TNF-alpha treatment. This study demonstrates that plaque NIRF imaging is feasible with a novel single chain antibody and that ED-B expression is closely associated with inflammation in experimental atherosclerosis.

A Chemical Proteomics Approach for the Identification of Accessible Antigens Expressed in Human Kidney Cancer

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

EDB fibronectin and angiogenesis – a novel mechanistic pathway

Extra domain-B containing fibronectin (EDB(+) FN), a recently proposed marker of angiogenesis, has been shown to be expressed in a number of human cancers and in ocular neovascularization in patients with proliferative diabetic retinopathy. To gain molecular understanding of the functional significance of EDB(+) FN, we have investigated possible regulatory mechanisms of induction and its role in endothelial cell proliferation and angiogenesis. Human vascular endothelial cells were cultured in high levels of glucose, and fibrogenic growth factors, transforming growth factor-beta1 (TGF-beta1) and endothelin-1 (ET-1). Our results show that high glucose levels, TGF-beta1, and ET-1 upregulated EDB(+) FN expression. Treatment of cells exposed to high glucose with TGF-beta1 neutralizing antibody and ET receptor antagonist prevented high glucose-induced EDB(+) FN expression. In order to elucidate the functional significance of EDB(+) FN upregulation, cells were subjected to in vitro proliferation and angiogenesis assays following EDB peptide treatment and specific EDB(+) FN gene silencing. Our results show that exposure of cells to EDB peptide increased vascular endothelial growth factor (VEGF) expression, endothelial proliferation, and tube formation. Furthermore, specific EDB(+) FN gene silencing prevented both basal and high glucose-induced VEGF expression and reduced the proliferative capacity of endothelial cells. In conclusion, these results indicate that EDB(+) FN is involved in endothelial cell proliferation and vascular morphogenesis, findings which may provide novel avenues for the development of anti-angiogenic therapies.

ED-B fibronectin as a target for antibody-based cancer treatments

Chemotherapeutic agents for the treatment of solid cancers do not discriminate between malignant and normal tissue, but rather depend on the increased proliferation of tumour cells versus benign cells. To reach therapeutically active concentrations in the tumour, large doses of these rather unspecific compounds have to be given to the patient, often resulting in severe side effects. Therefore, the goal of modern cancer research is the development of highly selective compounds which are able to discriminate between tumour tissue and normal tissue. One promising approach in this direction is antibody-mediated targeted cancer therapy which may either block an important receptor–ligand interaction or deliver a therapeutically active molecule to an otherwise nonfunctional target. A prereq-uisite for such an approach is the tumour-selective expression of the respective target structure. This review discusses extra domain-B fibronectin as a promising target which is associated with tumour angiogenesis and tumour growth for the development of novel antibody-mediated thera-pies.

EB-D fibronectin expression in squamous cell carcinoma of the head and neck

ED-B fibronectin (ED-B FN), a glycoprotein involved in cell adhesion and migration, is expressed in fetal and neoplastic tissues and absent in their normal counterparts. The aim of this study is to evaluate the expression of this glycoprotein in relation to the histological and clinical data and to determine whether it has a prognostic value in patients with head and neck squamous cell carcinoma (HNSCC). Ninety-five cases were assessed for ED-B FN expression using immunohistochemistry. Positive ED-B FN expression was significantly associated with tumor grade (p=0.06) and primary tumor site (p=0.02). The larynx was the tumor site associated with the least ED-B FN expression. In univariate analysis, there was no association with disease-free survival (p=0.48), but the mean time to progression was clearly shorter in tumors with positive ED-B FN expression than in those with negative expression (6 vs. 11 months). Patients having tumors expressing the ED-B FN had a trend to a significant lower overall survival in the multivariate analysis (p=0.06). Our study showed that ED-B FN expression might have a prognostic value in patients with HNSCC.

Potentiation of Osteogenesis and AngiogenesisIn Vivoafter Implantation of Hydroxyapatite Combined with Autogenous Growth Factors

Objective: Growth factors accelerate healing by stimulating matrix production and potentiating angiogenesis. In order to determine whether analogous effects of growth factors can be observed during the early phase of fracture healing, the use of hydroxyapatite (HA) combined with autogenous growth factors was investigated in the present study. For the acquisition of autogenous growth factors the platelet-rich plasma fraction was separated from blood by means of centrifugation. Design: For the experimental procedure two cylindrical defects were created within the intercondylar region of pig femoral condyles. The proximal defect was filled with HA, the distal with HA enriched with growth factors. Ten and twenty days after surgery the specimens were examined immunohistochemically and ultrastructurally. In order to study angio- and osteogenesis, antibodies to factor VIII, ED-A and, ED-B fibronectin, and to α smooth muscle actin were used. Results: Even after ten days there was a distinct increase of neovascularisation due to the application of growth factors as demonstrated with factor VIII and ED-B fibronectin antibodies. Moreover, granulation tissue formation was accelerated. After ten days intense labelling for α smooth muscle actin and ED-A fibronectin was observed only in combination with growth factors. Due to the effects of growth factors there is activation of fibroblasts to synthesise the ED-A fibronectin necessary for the induction of myofibroblasts by growth factors. Additionally, a few ED-B positive areas of woven bone including osteoblasts were identified ultrastructurally within the defect area after ten days of local treatment with growth factors. Moreover, the cells responsible for the accelerated degradation of the implant enriched with growth factors were identified ultrastructurally. Conclusion: Platelet growth factors added to biodegradable hydroxyapatite stimulate angio- and osteogenesis during bone defect healing.

Maternal plasma cellular fibronectin concentrations in normal and preeclamptic pregnancies: A longitudinal study for early prediction of preeclampsia

Objective: The purpose of this study was to examine cellular fibronectin levels throughout normotensive and preeclamptic pregnancies and to analyze its predictive value for the detection of preeclampsia within the second trimester of pregnancy. Study Design: Blood samples were collected at 4-week intervals from 378 healthy, nulliparous women who were recruited before 16 weeks of gestation. Preeclampsia developed in 26 patients; 52 normotensive control subjects were matched from the same cohort. Plasma samples were assayed for ED-B fibronectin by enzyme-linked immunosorbent assay. Trends were compared between groups. Predictive values were determined with the use of second trimester assessments. Results: In both groups, fibronectin levels rose as pregnancy advanced, but in women with preeclampsia, this increase was significantly higher (94.5% vs 31.8%; P =.006). Throughout pregnancy, patients with preeclampsia exhibited significantly higher fibronectin levels than did control subjects. As early as 9 to 12 weeks of gestation, a difference was established (preeclampsia, 3.72 ± 0.21; control, 2.94 ± 0.22 μg/mL [mean ± SEM]; P =.008). The best cutoff point and time interval to calculate predictive values were 3.8 μg/mL and 22 to 26 weeks of gestation, respectively. Sensitivity, specificity, and positive and negative predictive values were 73%, 87%, 29%, and 98%, respectively; the odds ratio was 16.1 (95% CI, 8.6-30.2). Conclusion: In women in whom clinical preeclampsia developed, endothelial damage seemed to be present since early gestation. Cellular fibronectin levels of ≥3.8 μg/mL within 22 to 26 weeks of gestation may help in the early detection of preeclampsia in healthy nulliparous women. (Am J Obstet Gynecol 2002;187:595-601.)