Hypoxia-induced down-regulation of microRNA-449a/b impairs control over targeted SERPINE1 (PAI-1) mRNA - a mechanism involved in SERPINE1 (PAI-1) overexpression.

After publication of our article 1, we realized the need for posting a correction note in order to prevent i) overinterpretation of some results by the readers and ii) concerns about potentially unintended misguidance by the authors.

Obliterative airway remodeling: molecular evidence for shared pathways in transplanted and native lungs.

Obliteration of the small airways is a largely unresolved challenge in pulmonary medicine. It represents either the irreversible cause of functional impairment or a morphologic disorder of limited importance in a multitude of diseases. Bronchiolitis obliterans is a key complication of lung transplantation. No predictive markers for the onset of obliterative remodeling are currently available. To further elucidate the molecular mechanisms of airway remodeling, compartment-specific expression patterns were analyzed in patients. For this purpose, remodeled and nonremodeled bronchioli were isolated from transplanted and nontransplanted lung explants using laser-assisted microdissection (n = 24). mRNA expression of 45 fibrosis-associated genes was measured using quantitative real-time RT-PCR. For 20 genes, protein expression was also analyzed by immunohistochemistry. Infiltrating cells were characterized at conventional histology and immunohistochemistry. Obliterative remodeling of the small airways in transplanted and nontransplanted lungs shared similar grades of chronic inflammation and pivotal fibrotic pathways such as transforming growth factor ß signaling and increased collagen expression. Bone morphogenetic protein and thrombospondin signaling, and also matrix metalloproteinases and tissue inhibitor of metalloproteinases, were primarily up-regulated in obliterative airway remodeling in nontransplanted lungs. In transplanted lungs, clinical remodeled bone morphogenetic protein but nonremodeled bronchioli were characterized by a concordant up-regulation of matrix metalloproteinase-9, RANTES, and tissue inhibitor of metalloproteinase-1. These distinct expression patterns warrant further investigation as potential markers of impending airway remodeling, especially for prospective longitudinal molecular profiling.

Expression of myelopoiesis-associated microRNA in bone marrow cells of atypical chronic myeloid leukaemia and chronic myelomonocytic leukaemia.

The microRNA/miR deregulation in BCR-ABL-negative myelodysplastic-myeloproliferative neoplasms (MDS/MPN) is not known. Myelopoiesis-associated miR-10a, miR-17-5p, miR-155, miR-223 and miR-424 were analysed by real-time polymerase chain reaction (PCR) in bone marrow cells of atypical chronic myeloid leukaemia (aCML, n = 7) and chronic myelomonocytic leukaemia (CMML, n = 8) and were compared to BCR-ABL-positive chronic myelogenous leukaemia (CML, n = 10) and non-neoplastic haematopoiesis (n = 10). Down-regulation of miR-10a was found in CMML but also in CML (each p < 0.05, versus controls). Overexpression of miR-424 was detected in aCML (p < 0.05, versus CML and controls). Despite different compositions of bone marrow cells, expression of myelopoiesis-associated microRNA shows mainly similar patterns in aCML and its main differential diagnosis CMML and does not allow discrimination of these two MDS/MPN entities. Therefore, the link of deregulated microRNA expression to disease-related phenotype and the underlying molecular mechanism are still unknown.

Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) - a megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia.

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm showing aberrant bone marrow remodeling with increased angiogenesis, progressive matrix accumulation, and fibrosis development. Thrombospondins (TSP) are factors sharing pro-fibrotic and anti-angiogenic properties, and have not been addressed in PMF before. We investigated the expression of TSP-1 and TSP-2 in PMF related to the stage of myelofibrosis (n = 51) and in individual follow-up biopsies by real-time PCR, immunohistochemistry, and confocal laser scanning microscopy (CLSM). TSP-1 was significantly overexpressed (p < 0.05) in all stages of PMF when compared to controls. Individual follow-up biopsies showed involvement of TSP-1 during progressive myelofibrosis. TSP-2 was barely detectable but 40% of cases with advanced myelofibrosis showed a strong expression. Megakaryocytes and interstitial proplatelet formations were shown to be the relevant source for TSP-1 in PMF. Stroma cells like endothelial cells and fibroblasts showed no TSP-1 labeling when double-immunofluorescence staining and CLSM were applied. Based on its dual function, TSP-1 in PMF is likely to be a mediator within a pro-fibrotic environment which discriminates from ET cases. On the other hand, TSP-1 is a factor acting (ineffectively) against exaggerated angiogenesis. Both features suggest TSP-1 to be a biomarker for monitoring a PMF-targeted therapy.

Aberrant proplatelet formation in chronic myeloproliferative neoplasms.

Proplatelets represent pseudopodia of megakaryocytes which extend into bone marrow sinuses to release platelets. Proplatelets were visualized by immunohistochemical and confocal microscopy. In trephines from essential thrombocythaemia (ET, n=10), fibrotic (n=10) and pre-fibrotic (n=10) primary myelofibrosis (PMF) there was a significant increase of proplatelet density compared with normal bone marrow samples (n=10; p<0.001). Manifest fibrosis exhibited the highest density and volume ratio with significant differences to non-fibrotic PMF (p<0.001) and ET (p<0.001). This study demonstrates that besides megakaryocytic proliferation extensive pseudopodial proplatelet formation provides a hallmark of MPN. Fibrosing differ from non-fibrosing MPN by density and size of aberrant proplatelets.

"Hypoxia-induced down-regulation of microRNA-449a/b impairs control over targeted SERPINE1 (PAI-1) mRNA - a mechanism involved in SERPINE1 (PAI-1) overexpression".

BACKGROUND: In damaged organs tissue repair and replacement of cells by connective tissue provokes a response of fibroblasts to cellular stress factors such as hypoxia.MicroRNAs (miRNA) are small non-coding RNA molecules which bind to their mRNA targets which eventually lead to repression of translation. Whether the response of fibroblasts to stress factors also involves the miRNA system is largely unknown. RESULTS: By miRNA profiling we identified down-regulation of miRNA-449a/b expression in hypoxic fibroblasts. Specific miRNA inhibitors and mimics showed direct evidence for targeting the serine protease inhibitor (serpin) protein (SERPINE1; plasminogen activator inhibitor-1, PAI-1) by miRNA-449a/b leading to SERPINE1 mRNA and protein up- and down-regulation, respectively. SERPINE1 expression in vivo could be located predominantly in areas of fibrosis and remodeling. CONCLUSIONS: Our study offers serious lines of evidence for a novel hypoxia-dependent mechanism involving hypoxia-induced decrease of clustered miRNA-449a/b, hypoxia-induced amplification of concomitant increase of targeted SERPINE1 (PAI-1) and its overexpression in tissues showing a hypoxic environment.

Identification of new target molecules PTK2, TGFBR2 and CD9 overexpressed during advanced bone marrow remodelling in primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by remodelling of the bone marrow, including progressive myelofibrosis and exaggerated angiogenesis. Advanced PMF frequently shows a full-blown fibre meshwork, which avoids aspiration of cells, and the expression profile of genes related to stroma pathology at this stage remains largely undetermined. We investigated bone marrow core biopsies in PMF showing various degrees of myelofibrosis by custom-made low density arrays (LDA) representing target genes with designated roles in synthesis of extracellular matrix, matrix remodelling, cellular adhesion and motility. Among a set of 11 genes up-regulated in advanced stages of PMF (P < or = 0.01) three candidates, PTK2 protein tyrosine kinase 2 (PTK2), transforming growth factor beta type II receptor (TGFBR2) and motility-related protein-1 (CD9 molecule, CD9), were investigated in more detail. PTK2, TGFBR2 and CD9 were significantly overexpressed in larger series of advanced PMF stages (P < or = 0.01 respectively). Endothelial cells of the increased microvessel network in PMF could be identified as a predominant source for PTK2, TGFBR2 and CD9. CD9 also strongly identified activated fibroblasts in advanced myelofibrosis. We conclude that PTK2, TGFBR2 and CD9 represent new target molecules involved in bone marrow remodelling of PMF and warrant further investigation for potential targeted therapy.

Genomic characterisation, chromosomal assignment and in vivo localisation of the canine high mobility group A1 (HMGA1) gene.

BACKGROUND: The high mobility group A1 proteins (HMGA1a/HMGA1b) are highly conserved between mammalian species and widely described as participating in various cellular processes. By inducing DNA conformation changes the HMGA1 proteins indirectly influence the binding of various transcription factors and therefore effect the transcription regulation. In humans chromosomal aberrations affecting the HMGA1 gene locus on HSA 6p21 were described to be the cause for various benign mesenchymal tumours while high titres of HMGA1 proteins were shown to be associated with the neoplastic potential of various types of cancer. Interestingly, the absence of HMGA1 proteins was shown to cause insulin resistance and diabetes in humans and mice. Due to the various similarities in biology and presentation of human and canine cancers the dog has joined the common rodent animal model for therapeutic and preclinical studies. Accordingly, the canine genome was sequenced completely twice but unfortunately this could not solve the structure of canine HMGA1 gene. RESULTS: Herein we report the characterisation of the genomic structure of the canine HMGA1 gene consisting of 7 exons and 6 introns spanning in total 9524 bp, the in vivo localisation of the HMGA1 protein to the nucleus, and a chromosomal assignment of the gene by FISH to CFA12q11. Additionally, we evaluated a described canine HMGA1 exon 6 SNP in 55 Dachshunds. CONCLUSION: The performed characterisations will make comparative analyses of aberrations affecting the human and canine gene and proteins possible, thereby providing a basis for revealing mechanisms involved in HMGA1 related pathogenesis in both species.