Siri Lehtonen

Overexpression of peroxiredoxins I, II, III, V, and VI in malignant mesothelioma

Peroxiredoxins (Prxs) are a recently characterized group of thiol‐containing proteins with efficient antioxidant capacity, capable of consuming hydrogen peroxide in living cells. Altogether six distinct Prxs have been characterized in mammalian tissues. Their expression was investigated in histological samples of mesothelioma and in cell lines established from the tumours of mesothelioma patients. Four cases with histopathologically healthy pleura from non‐smokers were used as controls. Healthy pleural mesothelium was negative or very weakly positive for all Prxs. In mesothelioma, the most prominent reactivity was observed with Prxs I, II, V, and VI. Prx I was highly or moderately expressed in 25/36 cases, the corresponding figures for Prxs II–VI being 27/36 (Prx II), 13/36 (Prx III), 2/36 (Prx IV), 24/36 (Prx V), and 30/36 (Prx VI). Positive staining was observed both in the cytosolic and the nuclear compartment, with the exception of Prx III, which showed no nuclear reactivity. The staining pattern of Prxs III and V was granular. Immunoelectron microscopic localization of Prxs was in accordance with the immunohistochemical findings, showing diffuse cytoplasmic localization for Prxs I, II, IV, and VI and distinct mitochondrial labelling for Prxs III and V. There was no significant association between the extent of staining and different Prxs. It appeared that Prxs may not have prognostic significance, but being prominently expressed in most mesotheliomas these proteins, at least in theory, may play a role in the primary drug resistance of this disease. Copyright

Peroxiredoxins, a novel protein family in lung cancer

Cigarette smoke, the major risk factor for lung cancer, induces an accumulation of reactive oxygen species. These have multiple effects on cell defense, cell proliferation and cell death. Thus, compounds involved in the regulators of redox balance can be hypothesized to play a fundamental role in both carcinogenesis and tumor progression. Here, we have evaluated the expressions of all 6 peroxiredoxins (Prxs I–VI) in lung carcinomas. Prxs represent a protein family with the capability of breaking down hydrogen peroxide; thus, they can participate in cellular antioxidant defense, regulate cell proliferation and increase drug resistance of cultured cells. Altogether 92 cases were investigated by immunohistochemistry, including 32 adenocarcinomas, 45 squamous cell, 9 small cell and 6 other carcinomas. Additionally, 11 cases with adenocarcinoma or squamous cell carcinoma were studied by Western analysis and/or by RT‐PCR. Prxs I, II, IV and VI were particularly elevated in lung carcinomas as assessed by immunohistochemistry and/or RT‐PCR. Western analysis revealed that Prxs I and IV were significantly elevated in tumors compared to nonmalignant tissue (p = 0.04 and 0.002, respectively). There were remarkable variations in Prx expression in various tumor subtypes, the most striking being Prx IV expression, which was mainly associated with adenocarcinoma. Elevated Prx VI expression was associated with high‐grade squamous cell carcinoma (p = 0.03) and Prx II expression, with advanced tumor stage (p = 0.01). Our results suggest that Prxs may have effects on the progression of lung cancer.

The Mechanism of Action of Induced Membranes in Bone Repair

BACKGROUND Inducement of foreign-body granulation tissue is a relatively novel therapeutic modality in bone repair. A two-stage bone reconstruction method, known as the Masquelet technique, combines inducement of a granulation tissue membrane and subsequent bone autografting as a biphasic technique allowing reconstruction of large bone defects. In light of their already well-characterized osteogenesis-improving capabilities in animals, we performed this translational study to investigate these membranes in patients. METHODS Fourteen patients with complicated fractures and bone defects were randomly selected for this study. Biopsy samples of foreign-body-induced membranes were collected at different time points during scheduled surgical procedures. The membranes were co-cultured with mesenchymal stromal cells, and differentiation into the osteoblastic lineage was assessed by measuring alkaline phosphatase activity, aminoterminal propeptide of type-I procollagen (PINP) production, and Ca2+ concentration. Histological characteristics were evaluated with image analysis. Quantitative reverse transcription polymerase chain reaction was used to measure vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and type-I collagen (Col-1) expression. RESULTS The induced membranes were characterized histologically by maturating vascularized fibrous tissue. The vascularization was greatest in one-month-old samples and decreased to <60% in three-month-old samples. One-month-old membrane samples had the highest expression of VEGF, IL-6, and Col-1, whereas two-month-old membranes expressed <40% of the levels of the one-month-old membranes. Specific alkaline phosphatase activity, PINP production, and Ca2+ concentration were increased in co-cultures when a membrane sample was present. In cultures of one-month-old membranes, PINP production was more than two times and Ca2+ deposition was four times higher than that in cultures of two-month-old membranes. CONCLUSIONS The induced membranes have osteogenesis-improving capabilities. These capabilities, however, appear to decrease over time. We speculate that the optimal time for performing second-stage surgery may be within a month after implantation of foreign material.

Cell Surface Structures Influence Lung Clearance Rate of Systemically Infused Mesenchymal Stromal Cells

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM‐MSCs) and umbilical cord blood (UCB‐MSCs). The BM‐MSCs and UCB‐MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB‐MSCs had higher expression levels of α4 integrin (CD49d, VLA‐4), α6 integrin (CD49f, VLA‐6), and the hepatocyte growth factor receptor (c‐Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells. STEM CELLS2013;31:317–326

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.

Toll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13

Human mesenchymal stem cells (hMSCs) are multipotent cells that are found in the bone marrow. Inflammation and tissue damage mobilize MSCs and induce their migration towards the damaged site through mechanisms that are not well defined. Toll-like receptor-9 (TLR9) is a cellular receptor for microbial and vertebrate DNA. Stimulation of TLR9 induces inflammatory and invasive responses in TLR9-expressing cells. We studied here the expression of TLR9 in human MSCs and the effects of synthetic TLR9-agonists on their invasion. Constitutive expression of TLR9 was detected in human MSCs but the expression was suppressed when MSCs were induced to differentiate into osteoblasts. Using standard invasion assays and a novel organotypic culture model based on human myoma tissue, we discovered that stimulation with the TLR9 agonistic, CpG oligonucleotides increased the invasion capacity of undifferentiated MSCs. Simultaneously, an increase in MMP-13 synthesis and activity was detected in the CpG-activated MSCs. Addition of anti-MMP-13 antibody significantly diminished the CpG-induced hMSC invasion. We conclude that treatment with TLR9-ligands increases MSC invasiveness, and this process is at least partially MMP-13-mediated.

Mitochondrial function and energy metabolism in umbilical cord blood- and bone marrow-derived mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are an attractive choice for a variety of cellular therapies. hMSCs can be isolated from many different tissues and possess unique mitochondrial properties that can be used to determine their differentiation potential. Mitochondrial properties may possibly be used as a quality measure of hMSC-based products. Accordingly, the present work focuses on the mitochondrial function of hMSCs from umbilical cord blood (UCBMSC) cells and bone marrow cells from donors younger than 18 years of age (BMMSC <18) and those more than 50 years of age (BMMSC >50). Changes of ultrastructure and energy metabolism during osteogenic differentiation in all hMSC types were studied in detail. Results show that despite similar surface antigen characteristics, the UCBMSCs had smaller cell surface area and possessed more abundant rough endoplasmic reticulum than BMMSC >50. BMMSC <18 were morphologically more UCBMSC-like. UCBMSC showed dramatically higher mitochondrial-to-cytoplasm area ratio and elevated superoxide and manganese superoxide dismutase (MnSOD) levels as compared with BMMSC >50 and BMMSC <18. All hMSCs types showed changes indicative of mitochondrial activation after 2 weeks of osteogenic differentiation, and the increase in mitochondrial-to-cytoplasm area ratio appears to be one of the first steps in the differentiation process. However, BMMSC >50 showed a lower level of mitochondrial maturation and differentiation capacity. UCBMSCs and BMMSCs also showed a different pattern of exocytosed proteins and glycoproteoglycansins. These results indicate that hMSCs with similar cell surface antigen expression have different mitochondrial and functional properties, suggesting different maturation levels and other significant biological variations of the hMSCs. Therefore, it appears that mitochondrial analysis presents useful characterization criteria for hMSCs intended for clinical use.

Divergent expression of claudin -1, -3, -4, -5 and -7 in developing human lung

BackgroundClaudins are the main components of tight junctions, structures which are associated with cell polarity and permeability. The aim of this study was to analyze the expression of claudins 1, 3, 4, 5, and 7 in developing human lung tissues from 12 to 40 weeks of gestation.Methods47 cases were analyzed by immunohistochemisty for claudins 1, 3, 4, 5 and 7. 23 cases were also investigated by quantitative RT-PCR for claudin-1, -3 and -4.ResultsClaudin-1 was expressed in epithelium of bronchi and large bronchioles from week 12 onwards but it was not detected in epithelium of developing alveoli. Claudin-3, -4 and -7 were strongly expressed in bronchial epithelium from week 12 to week 40, and they were also expressed in alveoli from week 16 to week 40. Claudin-5 was expressed strongly during all periods in endothelial cells. It was expressed also in epithelium of bronchi from week 12 to week 40, and in alveoli during the canalicular period. RT-PCR analyses revealed detectable amounts of RNAs for claudins 1, 3 and 4 in all cases studied.ConclusionClaudin-1, -3, -4, -5, and -7 are expressed in developing human lung from week 12 to week 40 with distinct locations and in divergent quantities. The expression of claudin-1 was restricted to the bronchial epithelium, whereas claudin-3, -4 and -7 were positive also in alveolar epithelium as well as in the bronchial epithelium. All claudins studied are linked to the development of airways, whereas claudin-3, -4, -5 and -7, but not claudin-1, are involved in the development of acinus and the differentiation of alveolar epithelial cells.

Inhibition of cytokine secretion from adipocytes by 1,25-dihydroxyvitamin D3 via the NF-κB pathway

Adipose tissue inflammation is an important pathological process in obese people, associated with diabetes and cardiovascular disease. We hypothesized that 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] inhibits cytokine secretion from adipocytes via direct inhibition of transcription factor nuclear factor‐κB (NF‐κB). We utilized two different human models. Bone marrow‐derived human mesenchymal stromal cells (hMSCs) differentiated into adipocytes, and adipocytes isolated from biopsies stimulated with lipopolysaccharide (LPS) were treated with or without 1,25(OH)2D3. Expression and secretion of interleukin‐6 (IL‐6) were measured by quantitative RT‐PCR analysis and ELISA. Assessment of NF‐κB nuclear translocation, DNA binding activity was performed by immunofluorescence (IF) and electrophoretic mobility assay (EMSA). Inhibitor κB (IκB) and its phosphorylation were detected by Western blot (WB) analysis. Simultaneous 1,25(OH)2D3 cotreatment significantly reduced LPS‐stimulated (10 ng/ml) IL‐6 secretion dose dependently by 15% at 10–10 M and 26% at 10–7 M (P<0.05) in hMSCs, while preincubation with 1,25(OH)2D3 (10–7 M) for 24 h reduced IL‐6 secretion by 24 and 35% (P<0.001) and mRNA levels by 34 and 30% (P<0.05) in hMSCs and isolated adipocytes, respectively. 1,25(OH)2D3 suppressed LPS‐stimulated IκB phosphorylation‐mediated NF‐κB translocation into the nucleus were evident from WB, IF, and EMSA. 1,25(OH)2D3 inhibits LPS‐stimulated IL‐6 secretion in two human adipocyte models via interference with NF‐κB signaling.—Mutt, S. J., Karhu, T., Lehtonen, S., Lehenkari, P., Carlberg, C., Saarnio, J., Sebert, S., Hyppönen, E., Järvelin, M.‐R., Herzig, K.‐H. Inhibition of cytokine secretion from adipocytes by 1,25‐dihydroxyvitamin D3 via the NF‐κB pathway. FASEB J. 26, 4400–4407 (2012). www.fasebj.org

CD200 Positive Human Mesenchymal Stem Cells Suppress TNF-Alpha Secretion from CD200 Receptor Positive Macrophage-Like Cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.