Diminished met signaling in podocytes contributes to the development of podocytopenia in transplant glomerulopathy.

Transplant glomerulopathy (TxG) can show secondary focal and segmental glomerulosclerosis (FSGS). FSGS in native kidneys is caused by podocytopenia. This study examines podocytopenia and the role of decreased paracrine Met activation on podocytes by decreased glomerular hepatocyte growth factor (HGF) levels in the development of podocytopenia in TxG. Podocytes were counted in 10 zero-hour biopsies and 10 specimens each with and without TxG. HGF/Met was examined with immunostains and quantitative RT-PCR in a set of three consecutive biopsies from 10 patients with TxG, including the diagnostic biopsy (DiagnBx) and the two previous biopsies (1stPrevBx and 2ndPrevBx). Antiapoptotic effects of HGF on podocytes were examined in vitro. Mean podocyte numbers per glomerulus were lower and glomerular volume higher in TxG. Fewer of the two preceding biopsies of the patients than of the controls contained phospho-Met(Tyr1349)-positive podocytes (2 of 8 versus 7 of 7, P = 0.0070; 4 of 9 versus 9 of 9, P = 0.0294). Glomerular HGF mRNA levels were lower in the 1stPrevBx of the patients (0.049 ± 0.083 versus 0.284 ± 0.331; P = 0.0155). In vitro, HGF stimulation of podocytes resulted in antiapoptotic phosphorylation of AKT and extracellular signal-regulated kinase (ERK) and induction of X-linked inhibitor of apoptosis protein (XIAP). Decreased antiapoptotic Met signaling in podocytes, probably due to decreased HGF secretion by glomerular epithelial cells, could contribute to podocyte loss and FSGS in TxG.

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.

Aberrant microRNA expression pattern in myelodysplastic bone marrow cells.

The microRNA/miR system might contribute to deregulation of cell homeostasis/disease phenotype. This is the first approach to generate an expression profile of 365 microRNAs in myelodysplastic syndromes (MDS) with normal karyotype (n=12) and distinct cytogenetic aberrations (n=12). In MDS-del(5q), a series of microRNAs not in the 5q-region was increased. MicroRNAs encoded on chromosomes 5, 7 and 8 were not differentially expressed in MDS with del(5q), -7 or +8. Evaluation in a larger cohort could confirm the up-regulation of the miR-1 in MDS. These findings provide evidence that MDS-haematopoiesis is distinct in its microRNA-expression pattern from non-neoplastic cells.

Significant inverse correlation of microRNA-150/MYB and microRNA-222/p27 in myelodysplastic syndrome.

We investigated whether, in myelodysplastic syndromes (MDS), aberrant expression of miR-150/miR-221/miR-222 and their designated target mRNA molecules MYB, p27 and c-KIT may be involved in insufficient haematopoiesis. In a series of MDS (n=52), an aberrant increase of miR-150 was found only in MDS with associated del(5q) (n=9; p<0.01). The mRNA expression of transcription factor MYB, the designated target of miR-150, was shown to correlate inversely with the miR-150 level. Acute leukaemia evolving from MDS (n=11) showed significantly decreased levels of miR-221 but not miR-222. We conclude that inhibition of proliferation via over-expressed miR-150 might contribute to myelodysplastic haematopoiesis in MDS-del(5q).

MicroRNA expression profiling of megakaryocytes in primary myelofibrosis and essential thrombocythemia.

In primary myelofibrosis (PMF) and essential thrombocythemia (ET) the megakaryocytic lineage characteristically shows aberrant proliferation and maturation in which the regulatory microRNA (miR) system might be involved. Laser-microdissected PMF and ET megakaryocytes were analysed with real-time polymerase chain reaction (PCR) low density arrays comprising 365 microRNAs. The highest megakaryocytic expression levels were observed for miR-223, which is known to be expressed also in granulopoiesis. Cluster analysis revealed a tendency of disease-specific megakaryocytic microRNA expression profiles indicating that a complex shift of microRNA expression appears to be the underlying defect. Increased accumulation of miR-146b was observed in cellular stage PMF (p = 0.0125) but not ET megakaryopoiesis. In conclusion, this is the first microRNA profiling of in situ-derived PMF, ET and normal megakaryocytes.

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.

JAK2(V617F) allele burden discriminates essential thrombocythemia from a subset of prefibrotic-stage primary myelofibrosis.

OBJECTIVE: Among Philadelphia chromosome-negative myeloproliferative neoplasms (Ph(-) MPN), essential thrombocythemia (ET) and the prefibrotic phase of primary myelofibrosis (PMF) represent two subtypes with considerable overlap. MATERIALS AND METHODS: In this study, histopathological classification of 490 MPN cases was correlated with the allelic burden of JAK2(V617F) and MPL(W515L). RESULTS: Ph(-) MPN entities largely overlap with regard to JAK2(V617F) and MPL(W515L) allele burden, but ET displayed mutant allele burden <50%. PMF with different stages of myelofibrosis all yielded similar JAK2(V617F) allele burden. At initial presentation one-quarter of prefibrotic PMF cases exhibited an allele burden exceeding 50% (38% median JAK2(V617F) alleles, n=102). In ET, its main differential diagnosis, not a single case was found with >40% JAK2(V617F) alleles (median, 24% JAK2(V617F) alleles; n=90; p<0.001). Increase in JAK2(V617F) alleles during follow-up could not be linked to fibrosis or blastic progression but was related to polycythemic transformation in ET. MPL(W515L) was found in 3% of ET and 8% of PMF, with a significantly higher percentage of mutated alleles in fibrotic than prefibrotic PMF (median, 78% MPL(W515L) alleles; p<0.05). CONCLUSION: Histopathological categories ET and prefibrotic PMF correlate with significant differences in mutant allelic burden of JAK2(V617F), but not of MPL(W515L) which, by contrast to JAK2(V617F), shows a higher percentage of mutated alleles in fibrotic than in prefibrotic cases. Thus, for Ph(-) MPN in which ET and prefibrotic PMF represent the most probable diagnoses, a JAK2(V617F) allele burden >50% favors a diagnosis of prefibrotic PMF.

Megakaryocytic expression of miRNA 10a, 17-5p, 20a and 126 in Philadelphia chromosome-negative myeloproliferative neoplasm.

Micro RNA (miRNA) are small non-coding RNA molecules which have a post-transcriptional inhibitory regulation function, e.g. in megakaryopoiesis. A characteristic of Philadelphia chromosome-negative myeloproliferative neoplasm (Ph(-) MPN) is the abundance of morphologically aberrant megakaryocytes. Based on previously published in vitro megakaryocytic differentiation assay data, we selected miRNA 10a, 17-5p, 20a and 126 and potential target proteins (HOXA1, RUNX1) for analysis of laser-microdissected bone marrow megakaryocytes from Ph(-) MPN and controls (n=66). Furthermore, we tested a potential influence of cytoreductive treatment on miRNA expression in bone marrow cells during the course of Ph(-) MPN (n=18). In summary, miRNA 17-5p, 20a and 126 are constitutively expressed in Ph(-) MPN megakaryopoiesis while low or absent miRNA 10a appeared to correlate with strong megakaryocytic HOXA1 protein expression. No association to thrombocytosis, JAK2(V617F) mutations or cytoreductive treatment (bone marrow cells) were observed.

Expression of adhesion factor CD239 in bone marrow cells in chronic myeloproliferative diseases.

Patients suffering from Philadelphia chromosome-negative chronic myeloproliferative disease (Ph(-) CMPD), such as polycythaemia vera (PV), are frequently JAK2(V617F)-mutated and have an elevated risk for thromboembolic complications. Recent data indicated that the molecular basis of JAK2(V617F) and thrombosis might be related to increased expression of CD239, the Lutheran blood group/basal cell adhesion molecule, in PV-derived red blood cells. The aim of this study was to clarify whether JAK2(V617F) PV with thromboembolism is characterised by CD239 overexpression. Leukocyte-derived JAK2(V617F) was detectable in bone marrow cells but also in liver and spleen explants and transplants after thrombosis (95% JAK2(V617F)-positive Ph(-) CMPD, n = 66). CD239 was expressed in a constitutive fashion in PV-derived bone marrow cells and thus a pro-thrombotic function does not seem to be mediated by increased CD239 expression alone.

Expression profiling of apoptosis-related genes in megakaryocytes: BNIP3 is downregulated in primary myelofibrosis.

OBJECTIVE: In order to identify factors involved in the aberrantly regulated apoptosis of megakaryocytes in primary myelofibrosis (PMF), the mRNA expression of human megakaryocytes in situ was quantified by real-time polymerase chain reaction low-density arrays. MATERIALS AND METHODS: The mRNA from 200 to 300 laser-microdissected megakaryocytes per case from PMF (n=22) and control (n=10) bone marrow was reverse-transcribed into cDNA by random priming and subsequently amplified by primer-specific cDNA amplification. The mRNA of corresponding total bone marrow cells was reverse-transcribed into cDNA without the following amplification. For relative mRNA quantification, custom-made TaqMan low-density arrays with a setup of 48 different genes were applied. In addition, methylation analysis and immunohistochemistry of a selected candidate gene were accomplished. RESULTS: A trend toward an overall downregulation of apoptosis-associated genes could be observed in megakaryocytes, whereas the total bone marrow cellularity exhibited an overall upregulation of these factors. Among several candidates with statistically significant deregulation BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and protein kinase C beta1 were shown to be the most aberrantly expressed genes. CONCLUSION: Apoptosis-related gene expression profiling of human megakaryocytes reveals a set of candidates, most notably BNIP3, indicating that the increase of megakaryocytes in myeloproliferative neoplasia might not only be the result of increased proliferation but also of disturbed apoptosis.

Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR.

The determination of marker genes and gene clusters involved in disease pathogenesis is increasingly contingent on high-throughput methods of gene expression profiling. However, the concurrently increasing application of mRNA from formalin-fixed and paraffin-embedded (FFPE) tissue archives, as well as cell-type-specific approaches by laser-assisted microdissection, frequently results in very small and degraded quantities of RNA. Therefore, a successful amplification of cell-type-specific mRNA targets from FFPE tissues becomes more and more essential. To optimize the hitherto limited technical options, we applied 3 commercial amplification kits on FFPE single cells. We thereby determined the approach of target-specific cDNA amplification as being notably appropriate for subsequent real-time polymerase chain reaction, as a constant decrease of CT values by 14 polymerase chain reaction cycles could be demonstrated.

Bone morphogenetic proteins are overexpressed in the bone marrow of primary myelofibrosis and are apparently induced by fibrogenic cytokines.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasia characterized by progressive deposition of extracellular matrix components in the bone marrow. The involvement of members of the bone morphogenetic protein (BMP) family in aberrant bone marrow matrix homeostasis in PMF has not yet been investigated. Therefore, we analyzed expression of BMP1, an activator of latent transforming growth factor beta-1 (TGFbeta-1) and processor of collagen precursors, and other BMPs in bone marrow from PMF patients and controls (n = 95). Expression of BMP1, BMP6, BMP7, and BMP-receptor 2 was significantly increased in advanced stages of myelofibrosis compared with controls (P < or = 0.01), and enhanced levels of BMP6 expression were already evident in prefibrotic stages of PMF. Immunohistochemistry showed that bone marrow stromal cells and megakaryocytes were the major cellular sources of BMP1 protein. Because TGFbeta-1 and basic fibroblast growth factor have been shown to be important in the development of myelofibrosis, we studied the induction of BMPs by these cytokines in cultured fibroblasts. Fibroblasts treated with TGFbeta-1 showed a pronounced up-regulation of BMP6, suggesting that stromal cells may be susceptible to BMP activation by cytokines with a proven role in the pathogenesis of PMF. We conclude that BMP family members may play an important role in the pathogenesis of myelofibrosis in PMF and are apparently induced by cytokines such as TGFbeta-1.

The expression levels of telomerase catalytic subunit hTERT and oncogenic MYC in essential thrombocythemia are affected by the molecular subtype.

The role of telomerase catalytic subunit hTERT in clonal malignancies including human leukemia is fundamental in overcoming cell senescence and enabling prolonged proliferation. One direct transcriptional activator of hTERT is the oncogene MYC which is known to be, in turn, activated by JAK2. To explore the relationship of telomerase, MYC and JAK2 in chronic myeloproliferative diseases, we investigated hTERT and MYC expression in bone marrow cells of essential thrombocythemia (ET) and polycythemia vera (PV). We could determine an up-regulation of MYC expression exclusively in JAK2(wt) ET, whereas hTERT expression was rather inconsistent across the groups. Interestingly, a significant correlation between MYC and hTERT expression could only be established in homozygous JAK2(V617F) PV and control cases. Thus, the functional link between MYC and hTERT seems to be impaired depending on the molecular ET subtype, which in turn may have implications on the phenotype and course of the disease.