Elisa Lappi-Blanco

Apoptotic Activity is Increased in the Newly Formed Fibromyxoid Connective Tissue in Bronchiolitis Obliterans Organizing Pneumonia

Abstract. Bronchiolitis obliterans organizing pneumonia (BOOP) and usual interstitial pneumonia (UIP) are clinically and histologically distinguishable interstitial lung diseases both sharing the presence of the newly formed fibromyxoid connective tissue as a histologic feature. In BOOP the newly formed connective tissue is susceptible to even complete reversal, but in UIP it participates in the remodeling of the pulmonary tissue. In this study we have tested the hypothesis that the apoptotic activity in the fibromyxoid lesions is higher in BOOP than in UIP. Apoptotic activity in cells of the newly formed connective tissue in BOOP and UIP was visualized by TUNEL. Apoptosis-regulating proteins bcl-2, mcl-1, and bax were visualized by immunohistochemistry. The number of the apoptotic events was given as an apoptotic index giving the percentage of the apoptotic cells and bodies of the total cell number. Our results show that the apoptotic activity is clearly higher in the fibromyxoid lesions of BOOP (mean, 0.7; S.D., ±0.51) compared with those of UIP (mean, 0.13; S.D., ±0.14, p < 0.003). The results thus suggest that apoptosis has an important role in the resolution process of the newly formed connective tissue in BOOP. The low level of apoptosis in UIP could, on the other hand, suggest that a decreased apoptosis in cells of the fibromyxoid stroma might pathogenetically relate to a decreased resolution of these lesions in UIP. No significant difference was found in the expression of bcl-2, mcl-1, and bax in BOOP and in UIP, suggesting that regulation of apoptosis might partly bypass the influence of these proteins.

VEGF and bFGF are highly expressed in intraluminal fibromyxoid lesions in bronchiolitis obliterans organizing pneumonia

Bronchiolitis obliterans organizing pneumonia (BOOP) and usual interstitial pneumonia (UIP) are fibrous pulmonary disorders in which new fibromyxoid connective tissue is formed in distal air spaces. In BOOP this tissue is susceptible to even complete reversal, but in UIP it participates in the remodelling of the interstitium. Our previous study showed that in BOOP this newly formed intraluminal tissue is more capillarized than in UIP. This paper studies the immunohistochemical expression of vascular endothelial growth factor (VEGF) and its receptors Flt‐1 and Flk‐1, and basic fibroblast growth factor (bFGF) in BOOP and UIP. It was hypothesized that there would be a difference in the expression of these angiogenic growth factors paralleling the difference in their capillarization. The results show that both VEGF and bFGF are widely expressed in BOOP and in UIP. It was shown with two different VEGF antibodies that its expression was more pronounced in the intraluminal fibromyxoid lesions in BOOP than in UIP (p<0.001 and 0.004, respectively). Similarly, bFGF also showed stronger espression in BOOP than in UIP (p<0.02) in these areas. The expression of Flt‐1 and Flk‐1 was in agreement with the expression of VEGF. It is concluded that the growth factors VEGF and bFGF may have an important role in the pathogenesis of BOOP and UIP; furthermore, their expression correlates with the angiogenic activity of the newly formed intraluminal fibromyxoid connective tissue in BOOP. Angiogenesis mediated by these growth factors may be one reason for the reversal of intraluminal fibromyxoid connective tissue in BOOP and for its good clinical outcome. Copyright

MRI-guided trephine biopsy and fine-needle aspiration in the diagnosis of bone lesions in low-field (0.23 T) MRI system using optical instrument tracking

Abstract. The purpose of this study was to evaluate the feasibility of MRI-guided bone biopsy with optical instrument tracking and evaluate advantage of combined fine-needle aspiration (FNA) with trephine biopsy. Twenty trephine bone biopsies and 13 FNAs were performed under MRI and CT guidance in 14 patients. Informed consent was obtained from all patients. The evaluation of diagnostic accuracy was achieved by comparing the histopathological and cytological diagnosis with current or final diagnosis made during 6-month clinical follow-up. A 0.23-T open MRI scanner with interventional tools (Outlook Proview, Marconi Medical Systems, Cleveland, Ohio) was used. A surface coil was used. For trephine biopsy MRI-compatible bone biopsy set (Daum medical, Schwerin, Germany) was used. The FNA was performed with MRI compatible 20-G needle (Cook, Bloomington, Ind.). The diagnostic accuracy of MRI-guided trephine biopsy was 95%. The FNA sample diagnosis concurred with the histological in 54%. Our results show that MRI guidance in bone biopsies is accurate and safe. It is comparable to CT-guided or open biopsy. The role of combined FNA with bone biopsies remains controversial.

Intraluminal fibromyxoid lesions in bronchiolitis obliterans organizing pneumonia are high capillarized

Bronchiolitis obliterans organizing pneumonia (BOOP) and usual interstitial pneumonia (UIP; ie, cryptogenic fibrosing alveolitis of mural type, CFA) are clinically and histologically distinguishable interstitial lung diseases. Both contain intraluminal lesions of newly formed fibromyxoid connective tissue. In BOOP, the fibromyxoid lesions are susceptible to complete reversal, but in UIP they are supposed to participate in the remodeling of the interstitium. Our hypothesis was that capillarization of the intraluminal fibromyxoid lesions is more frequent in BOOP compared with UIP. In this study, we stained diagnostic thoracoscopic or open lung biopsy specimens of patients with BOOP (n = 9) and UIP (n = 10) with antibodies against human laminin, von Willebrand factor, and CD34 to reveal the microvasculature of intraluminal fibromyxoid lesions. Our results show that in BOOP there is abundant capillarization in the newly formed intraluminal fibromyxoid lesions often reminiscent of granulation tissue. The mean number of capillaries per area unit (mm2) was 107 +/- SD 74 in samples stained for laminin, 103 +/- SD 46 for von Willebrand factor, and 63 +/- SD 36 for CD34. In marked contrast, in UIP, the corresponding accounts were significantly lower, being 14 +/- SD 15 for laminin (P < .003), 11 +/- SD 14 for von Willebrand factor (P < .001) and 6 +/- SD 6 for CD34 (P < .001). The intraobserver (P < .001) and interobserver correlations (P < .002) were highly significant, showing that our results are reproducible. We conclude that the content and nature of the newly formed intraluminal connective tissue, for example, in the form of vascular growth factors, are different in BOOP and in UIP, and this partly leads to the different clinical course of these diseases.

Divergence of tight and adherens junction factors in alveolar epithelium in pulmonary fibrosis.

It has been proposed that an epithelial injury may be one of the multiple primary events in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The aim of this study was to characterize the tight junction and adherens junction proteins in normal human lung, IPF, cryptogenic organizing pneumonia, and asbestosis. We determined the immunohistochemical cell-specific expression of tight junction proteins claudin-1, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-7, as well as 3 adherens junction proteins, E-cadherin, N-cadherin, and β-catenin. We further analyzed the expression of claudin-1, claudin-3, and claudin-4 and E-cadherin, N-cadherin, and β-catenin at the transcriptional level by quantitative real-time reverse transcriptase polymerase chain reaction. The expression levels of both tight junction and adherens junction proteins were elevated in regenerative alveolar epithelium in pulmonary fibrosis as compared with the expression of these proteins in normal alveolar epithelium. In particular, the expression levels of claudins-1 and claudin-3 were clearly elevated in all diseases. Furthermore, the amounts of adherens junction proteins messenger RNAs (mRNAs) were also all increased in pulmonary fibroses in comparison with healthy controls, with N-cadherin showing the greatest increase in mRNA levels in all diseases. However, the amounts of claudin-1, claudin-3, and claudin-4 mRNAs in fibrotic lung were similar to or even lower than those measured in the healthy controls. It is possible that the diminished capacity to produce claudin mRNAs may be one explanation for poor repair capacity of alveolar epithelial cells in IPF.

Tenascin-C and alpha-smooth muscle actin positive cells are increased in the large airways in patients with COPD

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by inflammation and remodeling of the lungs. This results in alterations in extracellular matrix (ECM) and structural changes leading to airflow obstruction. We studied the expression of tenascin-C (Tn-C) and alpha smooth muscle actin (α-SMA), which act as a marker of myofibroblasts, in large airways from COPD patients. Our aim was to elucidate whether this expression correlated with smoking or with disease development.MethodsBronchoscopy was performed on 20 COPD patients (mean age 56 years; range 39-61; FEV1/FVC < 70% and FEV1 median 53% (range 33-69) of predicted). Age and smoking matched smokers (S) without COPD (n = 13) and age matched non-smokers (NS) (n = 14) served as controls. Bronchial mucosal biopsies were analyzed by immunohistochemistry. The distribution of Tn-C expression was assessed and graded in three levels, and the number of spindle shaped cells staining positive for α-SMA were counted.ResultsBiopsies from COPD patients had more (P < 0.001) Tn-C expression than the two control groups. A significantly (P < 0.05) increased number of spindle shaped cells expressing α-SMA was observed in COPD patients compared with the controls. Smokers and nonsmokers did not differ in this respect. The expression of Tn-C correlated positively (P < 0.001) to the number of α-SMA positive cells.ConclusionsWe demonstrate increased expression of Tn-C and α-SMA positive cells in the large airways in COPD. This was not associated to smoking per se, but to the presence of airway obstruction. Our findings add new information regarding remodeling characteristics and highlight the large airways as a potential site for airways obstruction in COPD.

Epithelial N-cadherin and nuclear β-catenin are up-regulated during early development of human lung

BackgroundThe aim of this study was to analyze the cell-specific expression of E- and N-cadherin and β-catenin in developing human lung tissues from 12 to 40 weeks of gestation.MethodsFortyseven cases of developing human lung including pseudoglandular, canalicular, saccular and alveolar periods were analyzed by immunohistochemisty for E- and N-cadherin and β-catenin and twentyone cases were also investigated by RT-PCR for E- and N-cadherin and β-catenin. For identifying the lung cells, the sections were also stained with antibodies against thyroid transcription factor-1 (TTF-1) and caveolin-1. Normal adult lung tissue was used as a control.E-cadherin was strongly expressed in epithelium of bronchi and large bronchioles from week 12 onwards and it was also positive in alveoli in pretype II cells and type II cells. N-cadherin was present in most of the epithelial cells of bronchi and the largest bronchioles during the pseudo-glandular and canalicular periods. N-cadherin was not detected in epithelium of developing alveoli. β-catenin was strongly membrane-bound and positively expressed in bronchial epithelium from week 12 to week 40; it showed nuclear positivity in both developing airway epithelium and in the cells underneath the epithelium during pseudo-glandular period and to a lesser degree also in the canalicular period. β-catenin was positive in pretype II cells as well as in type I and type II pneumocytes within alveoli.RT-PCR analyses revealed detectable amounts of RNAs of E- and N-cadherin and β-catenin in all cases studied. The amounts of RNAs were higher in early stages of gestation.ConclusionsE-cadherin is widely expressed in bronchial and alveolar epithelial cells. N-cadherin exhibit extensive epithelial positivity in bronchial epithelial cells during early lung development. The presence of β-catenin was observed in several cell types with a distinct location in tissue and cells in various gestational stages, indicating that it possesses several roles during lung development. The expressions of protein and mRNAs of E- and N-cadherin and β-catenin were higher in early gestation compared to of the end. Moreover, the expressions of these factors were higher during the lung development than in the adult human lung.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

BMP-4 expression has prognostic significance in advanced serous ovarian carcinoma and is affected by cisplatin in OVCAR-3 cells

Several angiogenesis-promoting factors have prognostic significance in ovarian cancer. The objective of this study was to evaluate whether traditional chemotherapy affects angiogenesis-related factors in ovarian carcinoma and to assess the clinical significance of these effects. To screen for angiogenesis-related factors of possible relevance, OVCAR-3 and A2780 ovarian cancer cells were treated with IC50 doses of cisplatin (CDDP) or docetaxel, or with bevacizumab, and mRNA expression of several angiogenesis-related factors was analyzed. Thrombospondin-1 (TSP-1), bone morphogenetic protein-4 (BMP-4), endothelin-1, and placental growth factor-2 were statistically significantly induced by CDDP. At protein level, CDDP also induced hypoxia-inducible factor-1α but not vascular endothelial growth factor. In carcinoma samples taken before and after platinum-based neoadjuvant chemotherapy from 28 patients with advanced, high-grade serous ovarian carcinoma, CD105 and factors most induced by CDDP (TSP-1 and BMP-4) were analyzed by immunohistochemistry. Strong expression of BMP-4 before chemotherapy was an independent prognostic factor of longer progression-free time (p = 0.002) and overall survival (p = 0.02), but it was not associated with neovascularization (as evaluated by CD105). However, changes in BMP-4 expression in samples analyzed before and after chemotherapy (observed in 22/28 patients) were not associated with prognosis. TSP-1 expression was not associated with clinical parameters. Our results indicate that in serous ovarian carcinoma, BMP-4 has prognostic significance, which is not angiogenesis-related. We also show that CDDP induces several angiogenesis-related growth factors in vitro and future studies are warranted to clarify the clinical significance of this phenomenon.

Myofibroblast expression in airways and alveoli is affected by smoking and COPD

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD.Methodsα-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistochemical findings were correlated with clinical data. α-SMA protein was also analysed by Western blotting from fibroblastic cells cultured from peripheral lung of non-smokers, smokers without COPD and smokers with COPD.ResultsIn many cases, the endings of the detached alveolar walls were widened, the structures of which were named as widened alveolar tips. Widened alveolar tips contained α-SMA positive cells, which were obviously myofibroblasts. There were less alveolar tips containing positive cells for α-SMA in alveoli and α-SMA positive cells in bronchioles in smokers and in COPD compared to non-smokers. The quantity of α-SMA positive cells was increased in bronchi in COPD. Tn-C was elevated in bronchi in COPD and smokers’ lung. The α-SMA protein level was 1.43-fold higher in stromal cells cultured from non-smokers than in those of smokers.ConclusionsMyofibroblasts are localized variably in normal and diseased lung. This indicates that they have roles in both regeneration of lung and pathogenesis of COPD. The widened alveolar tips, these newly characterized histological structures, seemed to be the source of myofibroblasts at the alveolar level.