High NHLRC2 expression is associated with shortened survival in lung adenocarcinoma.

Background: Certain variants of NHL repeat (named after NCL-1, HT2A and LIN-41)-containing protein 2 (NHLRC2) gene have been linked to severe fibrotic interstitial lung disease in children. The aim of the current study was to evaluate the expression of NHLRC2 in lung cell and tissue samples from patients with lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Methods: The expression of NHLRC2 in lung tissue samples was studied by immunohistochemistry (102 ADC, 111 SCC), mRNA in situ hybridization (4 ADC, 3 SCC), and Western blot analysis (3 ADC, 2 SCC). The immunohistochemical NHLRC2 expression was measured by image analysis software and the percentage of NHLRC2-positive cancer cells was evaluated by semiquantitative analysis. The immunohistochemical results of NHLRC2 were compared with the clinical and histological characteristics of the patients. NHLRC2 protein levels in primary stromal and epithelial lung cancer cell lines were measured by Western blot analysis. Results: NHLRC2 was mainly expressed in cancer cells and inflammatory cells within the tumor. The NHLRC2 expression evaluated by image analysis method was significantly higher in ADC compared with that in SCC (P<0.001). High NHLRC2 expression was associated with reduced disease specific survival (P=0.002), overall survival (P=0.001), and high mitotic activity (P=0.042) in ADC. Additionally, the proportion of NHLRC2-positive cancer cells analyzed by the semiquantitative method was significantly higher in ADC than in SCC (P<0.001). Conclusions: NHLRC2 expression was higher in lung ADC than in SCC and its expression was associated with poor survival in ADC patients. Further studies are required to clarify the pathogenetic role of NHLRC2 in lung cancer.

NHLRC2 expression is increased in idiopathic pulmonary fibrosis.

BACKGROUND: Variants of NHL repeat-containing protein 2 (NHLRC2) have been associated with severe fibrotic interstitial lung disease in early childhood and NHLRC2 has been listed as a differentially expressed gene between rapidly and slowly progressing idiopathic pulmonary fibrosis (IPF) patients. However, its cell type-specific localization in human lung tissue is unknown. The aim of this study was to evaluate NHLRC2 mRNA and protein expression in different cell types of lung tissue samples and to investigate the effect of transforming growth factor (TGF)-ß1 exposure on NHLRC2 expression in vitro. METHODS: The NHLRC2 expression in lung tissue samples was studied by immunohistochemistry (50 IPF, 10 controls) and mRNA in situ hybridization (8 IPF, 3 controls). The immunohistochemical NHLRC2 expression was quantified with image analysis software and associated with the clinical and smoking data of the patients. NHLRC2 expression levels in primary stromal and small airway epithelial cell lines after exposure to TGF-ß1 was measured by quantitative reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: NHLRC2 expression was detected especially in bronchiolar epithelial cells, type II pneumocytes and macrophages in normal lung. In the lungs of IPF patients, NHLRC2 was mainly expressed in hyperplastic alveolar epithelial cells lining fibroblast foci and honeycombs. NHLRC2 expression assessed by image analysis was higher in IPF compared to controls (p < 0.001). Ever-smokers had more prominent NHLRC2 staining than non-smokers (p = 0.037) among IPF patients. TGF-ß1 exposure did not influence NHLRC2 levels in lung cell lines. CONCLUSIONS: NHLRC2 expression was higher in IPF compared to controls being widely expressed in type II pneumocytes, macrophages, bronchiolar epithelium, and hyperplastic alveolar epithelium. Additionally, its expression was not regulated by the exposure to TGF-ß1 in vitro. Further studies are needed to clarify the role of NHLRC2 in IPF.

Decline in Mast Cell Density During Diffuse Alveolar Damage in Idiopathic Pulmonary Fibrosis.

Mast cells (MCs) are known to be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), although their role in acute exacerbations of IPF has not been investigated. The aims of the study were to evaluate the numbers of MCs in fibrotic and non-fibrotic areas of lung tissue specimens of idiopathic pulmonary fibrosis (IPF) patients with or without an acute exacerbation of IPF, and to correlate the MC density with clinical parameters. MCs of IPF patients were quantified from surgical lung biopsy (SLB) specimens (n = 47) and lung tissue specimens taken at autopsy (n = 7). MC density was higher in the fibrotic areas of lung tissue compared with spared alveolar areas or in controls. Female gender, low diffusion capacity for carbon monoxide, diffuse alveolar damage, and smoking were associated with a low MC density. MC densities of fibrotic areas had declined significantly in five subjects in whom both SLB in the stable phase and autopsy after an acute exacerbation of IPF had been performed. There were no correlations of MC densities with survival time or future acute exacerbations. The MC density in fibrotic areas was associated with several clinical parameters. An acute exacerbation of IPF was associated with a significant decline in MC counts. Further investigations will be needed to clarify the role of these cells in IPF and in the pathogenesis of acute exacerbation as this may help to identify some potential targets for medical treatment for this serious disease.

Extracellular matrix proteins produced by stromal cells in idiopathic pulmonary fibrosis and lung adenocarcinoma.

Idiopathic pulmonary fibrosis (IPF) and lung cancer share common risk factors, epigenetic and genetic alterations, the activation of similar signaling pathways and poor survival. The aim of this study was to examine the gene expression profiles of stromal cells from patients with IPF and lung adenocarcinoma (ADC) as well as from normal lung. The gene expression levels of cultured stromal cells derived from non-smoking patients with ADC from the tumor (n = 4) and the corresponding normal lung (n = 4) as well as from patients with IPF (n = 4) were investigated with Affymetrix microarrays. The expression of collagen type IV alpha 1 chain, periostin as well as matrix metalloproteinase-1 and -3 in stromal cells and lung tissues were examined with quantitative real-time reverse transcriptase polymerase chain reaction and immunohistochemistry, respectively. Twenty genes were similarly up- or down-regulated in IPF and ADC compared to control, while most of the altered genes in IPF and ADC were differently expressed, including several extracellular matrix genes. Collagen type IV alpha 1 chain as well as matrix metalloproteinases-1 and -3 were differentially expressed in IPF compared to ADC. Periostin was up-regulated in both IPF and ADC in comparison to control. All studied factors were localized by immunohistochemistry in stromal cells within fibroblast foci in IPF and stroma of ADC. Despite the similarities found in gene expressions of IPF and ADC, several differences were also detected, suggesting that the molecular changes occurring in these two lung illnesses are somewhat different.

Smoking effect on the ultrastructural properties of cultured lung myofibroblasts.

This study aimed at an ultrastructural characterization of myofibroblasts cultured from different compartments of lung from never-smokers and smokers with or without COPD. In addition, we evaluated the expression of alpha smooth muscle actin (α-SMA), a marker for myofibroblasts, and contractile properties. Stromal cells cultured from central and corresponding peripheral or only from peripheral lung of never-smokers, smokers without COPD and COPD patients were analyzed by transmission electron microscopy (TEM), immunoelectron microscopy (IEM), Western analysis and/or by collagen gel contraction assay. TEM revealed that myofibroblasts cultured from smokers and COPD had less prominent intracellular actin filaments. We also examined fibronexus (FNX), which is a typical ultrastructural feature of myofibroblasts, and observed that patients with COPD more frequently had tandem-like FNX as compared to other samples. Western analysis showed that the samples derived from the central lung of never-smokers expressed higher levels of α-SMA than those of smokers and COPD patients. Cells from central lung were less contractile than those from peripheral lung. We conclude that myofibroblasts have variable ultrastructural and functional properties based on their localization in the lung and, moreover, these properties are affected by both smoking history and COPD.

Moderate hypothermia with remote ischaemic preconditioning improves cerebral protection compared to deep hypothermia: a study using a surviving porcine model.

OBJECTIVES: The optimal temperature management of hypothermic circulatory arrest is still controversial. Moderate hypothermia preserves cerebral autoregulation and shortens cardiopulmonary bypass (CPB) duration. However, moderate hypothermia alone has inferior organ protection to deep hypothermia, so adjuncts that increase the ischaemic tolerance are needed. Thus, we hypothesized that a combination of remote ischaemic preconditioning (RIPC) and moderate hypothermia would be superior to deep hypothermia alone. METHODS: Sixteen pigs were randomized to either RIPC or control groups (8 + 8). The RIPC group underwent 4 cycles of transient hind limb ischaemia. The RIPC group underwent cooling with CPB to 24°C, and the control group underwent cooling with CPB to 18°C, followed by a 30-min arrest period and subsequent rewarming to 36°C. Measurements of cerebral metabolism were made from sagittal sinus blood samples and common carotid artery blood flow. The permissible periods of hypothermic circulatory arrest were calculated based on these measurements. Neurological recovery was evaluated daily during a 7-day follow-up, and the brain was harvested for histopathological analysis. RESULTS: Six pigs in the RIPC group reached normal neurological function, but none in the control group reached normal neurological function (P = 0.007). The composite neurological score of all postoperative days was higher in the RIPC group than in the control group [55 (52-58) vs 45 (39-51), P = 0.026]. At 24°C, the estimated permissible periods of hypothermic circulatory arrest were 21 (17-25) min in the RIPC group and 11 (9-13) min in the control group (P = 0.007). CONCLUSIONS: RIPC combined with moderate hypothermia provides superior cerebral protection.

Women with polycystic ovary syndrome present with altered endometrial expression of stanniocalcin-1†.

Stanniocalcin-1 (STC-1) is a pro-survival factor that protects tissues against stressors, such as hypoxia and inflammation. STC-1 is co-expressed with the endometrial receptivity markers, and recently endometrial STC-1 was reported to be dysregulated in endometriosis, a condition linked with endometrial progesterone resistance and inflammation. These features are also common in the endometrium in women with polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. Given that women with PCOS present with subfertility, pregnancy complications, and increased risk for endometrial cancer, we investigated endometrial STC-1 expression in affected women. Endometrial biopsy samples were obtained from women with PCOS and controls, including samples from overweight/obese women with PCOS before and after a 3-month lifestyle intervention. A total of 98 PCOS and 85 control samples were used in immunohistochemistry, reverse-transcription polymerase chain reaction, or in vitro cell culture. STC-1 expression was analyzed at different cycle phases and in endometrial stromal cells (eSCs) after steroid hormone exposure. The eSCs were also challenged with 8-bromo-cAMP and hypoxia for STC-1 expression. The findings indicate that STC-1 expression is not steroid hormone mediated although secretory-phase STC-1 expression was blunted in PCOS. Lower expression seems to be related to attenuated STC-1 response to stressors in PCOS eSCs, shown as downregulation of protein kinase A activity. The 3-month lifestyle intervention did not restore STC-1 expression in PCOS endometrium. More studies are warranted to further elucidate the mechanisms behind the altered endometrial STC-1 expression and rescue mechanism in the PCOS endometrium.

Safety of direct true lumen cannulation after venous exsanguination: a study in a surviving porcine model†.

OBJECTIVES: Type A aortic dissection requires immediate surgery. Traditional cannulation methods such as the central aortic cannulation with the Seldinger technique and axillary cannulation are primary choices. Yet in the presence of tamponade or severe cardiogenic shock, these can be too time-consuming to complete. Direct true lumen cannulation after venous exsanguination not only avoids this issue but also leads to transient global ischaemia. We studied the safety of direct true lumen cannulation from the aspect of global ischaemia in a surviving porcine model. METHODS: Twelve pigs were randomized to either control or intervention groups (6 + 6). The intervention group underwent simulated direct true lumen cannulation by exsanguination and circulatory arrest for 5 min at 35°C before cardiopulmonary bypass (CPB). Both groups underwent CPB cooling to 25°C followed by a 25-min arrest period and subsequent warming to 36°C. Neuron-specific enolase levels were measured at 6 time-points from blood samples. Near-infrared spectroscopy was used to determine brain oxygenation. The neurological recovery was evaluated daily during a 7-day follow-up, and the brain was harvested for a histopathological analysis after euthanization. RESULTS: All pigs recovered their normal neurological behaviour. The neurobehavioural total score on postoperative day 2 reached borderline statistical significance, thus favouring the intervention group [(9 (8.75-9) vs 6.5 (5.5-9), P = 0.06]. Near-infrared spectroscopy values and neuron-specific enolase levels slightly favoured the control group during the cooling period, but the difference was not clinically significant. The histopathological analysis showed no difference between the groups. CONCLUSIONS: A 5-min period of normothermic global ischaemia before CPB does not impair the neurological outcome following hypothermic circulatory arrest in a surviving porcine model.

Biallelic mutations in human NHLRC2 enhance myofibroblast differentiation in FINCA disease.

The development of tissue fibrosis is complex and at the present time, not fully understood. Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA disease) have been described in patients with mutations in NHL repeat-containing protein 2 (NHLRC2). However, the molecular functions of NHLRC2 are uncharacterized. Herein, we identified putative interacting partners for NHLRC2 using proximity-labeling mass spectrometry. We also investigated the function of NHLRC2 using immortalized cells cultured from skin biopsies of FINCA patients and normal fibroblasts with NHLRC2 knock-down and NHLRC2 overexpressing gene modifications. Transmission electron microscopy analysis of immortalized cell cultures from three FINCA patients demonstrated multilamellar bodies and distinctly organized vimentin filaments. Additionally, two of three cultures derived from patient skin biopsies contained cells that exhibited features characteristic of myofibroblasts. Altogether, the data presented in this study show for the first time that NHLRC2 is involved in cellular organization through regulation of the cytoskeleton and vesicle transport. We conclude that compound heterozygous p.Asp148Tyr and p.Arg201GlyfsTer6 mutations in NHLRC2 lead to severe tissue fibrosis in humans by enhancing the differentiation of fibroblasts to myofibroblasts.

Niche matters: The comparison between bone marrow stem cells and endometrial stem cells and stromal fibroblasts reveal distinct migration and cytokine profiles in response to inflammatory stimulus.

OBJECTIVE: Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs) have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs) and endometrial fibroblasts (eSFs). MATERIALS AND METHODS: The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS)-induced state. RESULTS: Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF)-A, stromal cell-derived factor-1 alpha (SDF)-1α, interleukin-1 receptor antagonist (IL-1RA), IL-6, interferon-gamma inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs. CONCLUSION: Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation process towards stromal fibroblasts seemed to result in loss of stem cell surface markers, minimal migration activity and a subtler cytokine profile likely contributing to normal endometrial function.

Mucin-1 correlates with survival, smoking status, and growth patterns in lung adenocarcinoma.

Mucin-1 (MUC1) affects cancer progression in lung adenocarcinoma, and its aberrant expression pattern has been correlated with poor tumor differentiation and impaired prognosis. In this study, the immunohistochemical expression of MUC1 and Mucin-4 (MUC4) was analyzed in a series of 106 surgically operated stage I-IV pulmonary adenocarcinomas. MUC1 immunohistochemistry was evaluated according to the Nagai classification, and the immunohistochemical profile of the tumors was correlated with detailed clinical and histological data. The effect of cigarette smoke on MUC1 expression in lung cancer cell lines was examined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoelectron microscopy (IEM). In contrast to the normal apical localization of MUC1, a basolateral and cytoplasmic (depolarized) MUC1 expression pattern was frequently encountered in the high-grade subtypes, i.e., solid predominant adenocarcinoma and the cribriform variant of acinar predominant adenocarcinoma (p < 0.001), and was rarely observed in tumors containing a non-predominant lepidic component (p < 0.001). Furthermore, the altered staining pattern of MUC1 correlated with stage (p = 0.002), reduced overall survival (p = 0.031), and was associated with smoking (p < 0.001). When H1650 adenocarcinoma cells were exposed to cigarette smoke and analyzed by RT-qPCR and IEM, the levels of the MUC1 transcript and protein were elevated (p = 0.042). In conclusion, MUC1 participates in the pathogenesis of lung adenocarcinoma and associates with smoking both in vitro and in vivo. In lung adenocarcinoma, depolarized MUC1 protein expression correlated with histological growth patterns, stage, and patient outcome.

Iron-labeled adipose stem cells and neovascularization in rabbit calvarial critical-sized defects.

OBJECTIVE: The aim of this study was to evaluate the presence of iron-labeled adipose stem cells at the 2-week time point and vascular changes at the 2-week and 6-week time points using two different types of scaffolds. STUDY DESIGN: This study included 22 White New Zealand adult male rabbits. In six rabbits, full-thickness calvarial critical-sized defects were filled with autogenous adipose stem cells labeled with iron oxide seeded onto two scaffolds, namely, solid bioactive glass (BAG) or porous tricalcium phosphate granules (TCP) used on reciprocal sides of the skull. Eleven rabbits were implanted with adipose stem cell-seeded scaffolds without iron labeling for analysis of vascular changes. Five defects were left empty as negative control defects. The specimens were analyzed histologically at the 2-week and 6-week time points. RESULTS: The TCP group showed significantly more vascularity compared with the BAG group. A greater number of labeled stem cells were identified in the TCP group compared with the BAG group, but the difference was not statistically significant. CONCLUSIONS: This study revealed the differences in stem cell distribution and revascularization of the calvarial defect, which may be biomaterial dependent.

Pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an incurable lung disease with a poor prognosis. Fibroblasts and myofibroblasts are the key cells in the fibrotic process. Recently two drugs, pirfenidone and nintedanib, were approved for clinical use as they are able to slow down the disease progression. The mechanisms by which these two drugs act in in vitro cell systems are not known. The aim of this study was therefore to examine the effects of pirfenidone and nintedanib on fibroblasts and myofibroblasts structure and function established from patients with or without IPF. METHODS: Stromal cells were collected and cultured from control lung (n = 4) or IPF (n = 7). The cells were treated with pirfenidone and/or nintedanib and the effect of treatment was evaluated by measuring cell proliferation, alpha smooth muscle actin (α-SMA) and fibronectin expression by Western analysis and/or immunoelectron microscopy, ultrastructural properties by transmission electron microscopy and functional properties by collagen gel contraction and invasion assays. RESULTS: Both pirfenidone and nintedanib reduced in vitro proliferation of fibroblastic cells in a dose dependent manner. The number of cells from control lung was reduced to 47 % (p = 0.04) and of IPF cells to 42 % (p = 0.04) by 1 mM pirfenidone and correspondingly to 67 % (p = 0.04) and 68 % (p = 0.04), by 1 µM nintedanib. If both drugs were used together, a further reduced proliferation was observed. Both pirfenidone and nintedanib were able to reduce the amount of α-SMA and the myofibroblastic appearance although the level of reduction was cell line dependent. In functional assays, the effect of both drugs was also variable. CONCLUSIONS: We conclude that the ultrastructure and function of fibroblasts and myofibroblasts are affected by pirfenidone and nintedanib. Combination of the drugs reduced cell proliferation more than either of them individually. Human lung derived cell culture systems represent a potential platform for screening and testing drugs for fibrotic diseases.

High number of transplanted stem cells improves myocardial recovery after AMI in a porcine model.

OBJECTIVE: The clinical data considering the bone marrow mononuclear cell (BMMNC) therapy in treatment for acute myocardial infarction (AMI) are controversial and the mechanisms remain unknown. Our objective was to study the cardiac function and changes in cytokine levels after administration of BMMNC in experimental AMI model. DESIGN: Unlabeled or Super-Paramagnetic-Iron-Oxide-labeled BMMNCs or saline was injected into myocardium of 31 pigs after circumflex artery occlusion. Ejection fraction (EF) was measured preoperatively, postoperatively and at 21 days by echocardiography. Cardiac MRI was performed postoperatively and after 21 days in 7 BMMNC animals. Serum cytokine levels were measured at baseline, 24 h and 21 days. Cellular homing was evaluated comparing MRI and histology. RESULTS: From baseline to 21 days EF decreased less in BMMNC group (EF mean control -19 SD 12 vs. BMMNC -4 SD 15 percentage points p = 0.02). Cytokine concentrations showed high variability between the animals. MRI correlated with histology in cell detection and revealed BMMNCs in the infarction area. By MRI, EF improved 11 percentage points. The improvement in EF was associated with the number of transplanted BMMNCs detected in the myocardium. CONCLUSION: BMMNC injection after AMI improved cardiac function. Quantity of transplanted BMMNCs correlated with the improvement in cardiac function after AMI.

Mesenchymal stromal cells from female donors enhance breast cancer cell proliferation in vitro.

The interplay between tumor stroma and breast cancer cells (BCCs) is thought to play a significant role in breast cancer. The current knowledge of human mesenchymal stromal cell (MSC) and BCC interaction is contradictory, and the donor sex issue is not addressed at all. We hypothesized that donor sex could have an effect on proliferation of MSCs or BCCs in co-culture in vitro. Three estrogen receptor-negative BCC lines, 19 primary human MSCs and breast tissue-derived fibroblasts from 4 donors were used. MSCs from female donors enhanced BCC proliferation (p = 0.005). The change in BCC proliferation was only partly due to soluble factors excreted by MSCs. The highly aggressive BCC line MDA-MB- 231 induced the proliferation of MSCs (p < 0.001) and fibroblasts (p = 0.037) in co-culture experiments. The magnitude in proliferation change was cell line dependent and partly sex dependent.

Slug is associated with poor survival in squamous cell carcinoma of the lung.

We investigated the expression of slug in a large set of lung squamous and adenocarcinomas to determine common or dissimilar features in its expression in these two most common forms of lung cancer. To investigate slug related tumor spread we studied the expression of vimentin, claudin 1, MMP2 and MMP9 in these tumors and their relation to slug. Addition, cell invasion assays, mRNA analysis and zymographic tests were performed to study epitheliomesenchymal transition (EMT) related changes in slug blocked lung cell lines. According to the results slug expression did not significantly differ between squamous (SCC) and adenocarcinoma (AC) (P = 0.25). In SCC, slug associated with vimentin (P = 0.016). In AC, claudin 1 associated with MMP2 (P = 0.037). In SCC slug expression had a poor prognositic significance (P = 0.006) and it had independent prognostic value (P = 0.037). In AC MMP2 had a worsening impact on survival (P = 0.021) and it had independent prognostic value (P = 0.002). In cell invasion assays, slug knockdown inhibited the invasion and migration of BEAS-2B, SK-LU1 and SK-MES1 cell lines. The mRNA expression of claudin 1 was downregulated in SK-LU1 cell line. Both tumor cell lines expressed MMP2 and in SK-MES1 slug inhibited line MMP2 appeared to decrease. The results show that slug associated EMT is more pronounced in lung SCC than AC. Slug associated with vimentin in SCC and had an independent prognostic value in this tumor type. Forced slug inhibition might be one putative way of treatment of SCC of the lung.

Stromal cells can be cultured and characterized from diagnostic bronchoalveolar fluid samples obtained from patients with various types of interstitial lung diseases.

Increased proliferation of stromal cells is a typical feature encountered in several lung diseases. The objective of this study was to evaluate the success of standardized process for culturing stromal cells from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples collected from various patients and to characterize the cultured cells. Small volumes (average 15 mL) of BAL fluid samples were collected from 98 patients who underwent bronchoscopy and BAL for diagnostic purposes. The cells were cultured in vitro and characterized by immunohistochemistry, electron microscopy, flow cytometry and differentiation tests. Cells could be cultured from 62% of samples with the success rate varying with the disease (p = 0.003). Cultures from samples of the patients with idiopathic pulmonary fibrosis, non-specific interstitial pneumonia, connective tissue disorder associated interstitial lung disease and allergic alveolitis had a higher success rate than samples derived from control lung (p < 0.001, 0.03, 0.03 and 0.044, respectively). Smokers had a higher success rate compared with non-smokers (p = 0.035). The cultured cells were fibroblasts or myofibroblasts, but shared also similarities with progenitor-type cells. The study shows that mesenchymal cells can be cultured and studied from small volumes of diagnostic BAL fluid samples from patients with several different types of lung diseases.

Lung cancer-associated myofibroblasts reveal distinctive ultrastructure and function.

BACKGROUND: Cancer-associated stromal cells interact with carcinoma cells and thus participate in tumor growth. Our aim was to characterize the ultrastructure and contractile properties of stromal cells in collagen gel cultured from lung cancer of various histological types and from tumor-free lung. METHODS: Cells cultured from lung cancer (13 adenocarcinomas, six squamous cell carcinomas, one adenosquamous carcinoma, and one pleomorphic carcinoma) and tumor-free lung were analyzed by transmission electron microscopy and three-dimensional collagen gel contraction assays. The expression of α-smooth muscle actin (α-SMA), a recognized myofibroblast marker, was examined by immunoelectron microscopy from individual cells and by Western blotting from the whole cultured cell population. RESULTS: According to their ultrastructure, the cell lines were composed of fibroblastic and myofibroblastic cells. In electron microscopy, cells of lung cancer exhibited more myofibroblastic features displaying higher amounts of actin belts (p = 0.057) and α-SMA labeling (p = 0.010) than cells from tumor-free lung. Myofibroblasts cultured from lung cancer of smokers expressed less α-SMA labeling (p = 0.013) than counterparts from nonsmokers. Western blotting revealed that cancer-associated fibroblasts expressed more α-SMA (p = 0.006) than cells from tumor-free lung, whereas cells from tumor-free central lung of smokers showed less α-SMA (p = 0.039) than counterparts from nonsmokers. Cells cultured from cancer contracted more in collagen gel than those from tumor-free lung. The contractile capacity in collagen gel correlated with the frequency of extracellular component of fibronexus by transmission electron microscopy. CONCLUSIONS: Lung cancer-associated myofibroblasts are different both ultrastructurally and functionally when compared with cells cultured from tumor-free lung. Smoking altered myofibroblastic phenotype, regardless of their origin.

Analysis of molecular changes after autologous cell therapy in swine myocardial infarction tissue can reveal novel targets for future therapy.

Although several studies have demonstrated a functional recovery of infarcted myocardial tissue after cell therapy, little is known about the molecular mechanisms behind it. The aim of this study was to characterize the effect of cell therapy at the molecular level to screen for novel target candidates for future therapy of infarcted myocardial tissue. We used a swine acute myocardial infarction model evoked by transient occlusion of the circumflex coronary artery. Autologous bone marrow-derived mononuclear cells (BMMCs) or saline were injected intramyocardially or into the circumflex coronary artery. Samples for protein and RNA analysis were collected from the infarction area and healthy myocardium after a 3 week recovery period and analysed by two-dimensional gel electrophoresis (2DE) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Proteomic screening detected 13 protein spots which were altered after infarction but had been restored by BMMC treatment. The identification of seven proteins by mass spectrometry revealed that five proteins with decreased expression after infarction corresponded to mitochondrial proteins involved in energy metabolism. Their restored levels after BMMC treatment indicate their involvement in the recovery of heart function. In contrast, the elevated levels of α-crystallin B chain and cathepsin D after infarction suggest an involvement in the pathological mechanisms causing a decreased heart function. This study reveals that cell therapy with BMMCs after myocardial infarction causes restoration of several altered protein levels after 3 weeks and identifies potential marker proteins involved in the pathology of infarction.

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

BACKGROUND: Chronic 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. METHODS: α-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. RESULTS: In 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. CONCLUSIONS: Myofibroblasts 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.