点击显示 收起
【摘要】
Bone marrow fibrosis in chronic idiopathic myelofibrosis (cIMF) most likely represents an imbalance between synthesis and turnover of collagen fibers. Because the JAK-STAT signaling pathway is involved in the regulation of genes encoding matrix metalloproteinases (MMPs), we examined the expression of MMPs, their tissue inhibitors (TIMPs), and collagen types in relation to the JAK2 status (V617F mutation versus wild-type) in cIMF (n = 64). Whereas no correlation was found between the JAK2 status and MMP gene products, there was an evident association with the stage of disease. Membrane type 1-MMP (MMP-14) was overexpressed by up to 80-fold in advanced stages that progressed to fibrosis (P < 0.001), and megakaryocytes and endothelial cells were unmasked as the major cellular source. By contrast, a significantly higher expression of neutrophil collagenase (MMP-8) was encountered in the prefibrotic stages of cIMF (P < 0.001). Altogether, the stepwise progress of myelofibrosis in cIMF was associated with expression of a defined subset of target genes as shown in sequential trephine biopsies of cIMF patients. We conclude that the expression of matrix-modeling genes in cIMF is not influenced by the JAK2 mutation status but is predominantly related to the stage of disease.
--------------------------------------------------------------------------------
According to the World Health Organization classification, Philadelphia chromosome-negative chronic myeloproliferative disorders comprise polycythemia vera, essential thrombocythemia, and chronic idiopathic myelofibrosis (cIMF) along with more rare entities such as chronic neutrophilic leukemia and chronic eosinophilic leukemia/hypereosinophilic syndrome.1 cIMF shows progressive bone marrow fibrosis, increased angiogenesis, enhanced trafficking of CD34+ cells with extramedullary hematopoiesis and greater risk for transformation into acute leukemia.2 In up to 50% of patients, mutated and therefore constitutively activated JAK2 has been demonstrated to be the underlying molecular defect responsible for autonomous proliferation.3-7
The extracellular matrix (ECM) is a complex meshwork comprising various types of collagens, laminin, entactin, heparan sulfate, and proteoglycans.8 The ECM is substantially involved in the interaction of hematopoietic cells and bone marrow stroma cells.9 In normal bone marrow, resident fibroblasts are responsible for constitutively synthesized collagens.10 Patients with cIMF either present with manifest myelofibrosis or present with a prefibrotic, cellular phase with no or only minute degrees of collagen fiber increase in the bone marrow.11,12 The onset of progressive deposition of collagens in prefibrotic cIMF is variable. Collagen type-3 (COL-3, also termed reticulin) and collagen type-1 (COL-1), synthesized by nonclonal fibroblasts, seem to be the predominant ECM components in the process of myelofibrosis.2
The family of matrix metalloproteinases (MMPs) is comprised of more than 20 enzymes that can collectively degrade all components of the ECM.13 MMP-1, -8, -13, and -14 (MT1-MMP) are thought to be the most potent collagenolytic MMPs.13 Of note, MMP-1, -8, and -13 cleave the triple helix of collagens, thereby allowing the chains to unwind and become susceptible to further degradation by other MMPs. MMP-2 is a gelatinase but, in contrast to MMP-9, is also highly potent in the degradation of collagen types I, II, and III.13 It appears likely that an imbalance of ECM synthesis and activation of proteolytic enzymes paves the way for the increasing collagen meshwork in cIMF. Conversely, stem cell trafficking and angiogenesis as hallmarks in advanced cIMF stages require local matrix turnover, ie, proteolytic action, for full establishment.14 It has been demonstrated that the JAK-STAT pathway is involved in the activation of genes responsible for synthesis and secretion of MMPs.15,16 Besides revealing the role of JAKs and MMP activity in the physiological processes of hematopoiesis and differentiation, recruitment of this particular pathway has also been demonstrated during tumor invasion and neoangiogenesis.17-20 We tested the hypothesis of a potential correlation of an underlying JAK2 mutation in cIMF that leads to aberrant expression of MMPs, TIMPs, and COLs in different disease stages and during the course of the disease.
【关键词】 aberrant collagenase expression idiopathic myelofibrosis mutation
Materials and Methods
Bone Marrow Study Group
Formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines with proven cIMF were retrieved from the bone marrow registry of the Institute of Pathology, Hannover Medical School. Bone marrow trephines were routinely fixed in a solution containing phosphate-buffered formalin (pH 7.4) for 24 hours. The decalcification step was performed in an ethylenediaminetetraacetic acid-based solution (pH 7.5) for up to 48 hours. The study group (n = 88) comprised cellular, prefibrotic cIMF (n = 31), advanced cIMF with manifest myelofibrosis (n = 33), and 24 control cases showing normal hematopoiesis. Based on the World Health Organization classification and in close agreement with clinical data and presentation, patient bone marrow trephines were initially diagnosed to have a cIMF in the years 2000 to 2004. In addition, sequential bone marrow trephines in two patients were investigated, representing follow-ups of 3 years. cIMF cases were re-evaluated and subdivided into two groups depending on the degree of myelofibrosis after silver impregnation (Gomori) as described.11,21 For a summary of patient clinical data, see Table 1 .
Table 1. Brief Clinical Description of the Study Group
Real-Time Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR)
As we previously described,22 total RNA was extracted from total FFPE bone marrow cells after guanidinium isothiocyanate/proteinase K-based digestion and conventional organic extraction using phenol/chloroform. Total RNA (1 µg), pretreated with RNase-free (RNaseC) DNase (1 U/µg RNA, RQ1; Promega, Madison, WI), was transcribed into the complementary DNA using 500 ng of random hexamers (Amersham Pharmacia, Piscataway, NJ) and 200 U of SuperScript II RNaseC reverse transcriptase (Invitrogen, Karlsruhe, Germany) in a volume of 20 µl following the manufacture??s protocol. Negative controls were performed by using water instead of reverse transcriptase. Real-time PCR was performed on an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA). For sequences of PCR primers and TaqMan probes that amplify COL-1, COL-3, MMP-2, MMP-8, MMP-13, MT1-MMP, TIMP-1, TIMP-2, and the housekeeping gene ß-glucuronidase see Table 2 . The real-time PCR amplification was performed in a final reaction volume of 25 µl containing primers (250 nmol/L each), probe (150 nmol/L), 0.5 U of Platinum Taq polymerase (Invitrogen), 200 µmol/L each of dATP, dCTP, dTTP, and dGTP in 1x Platinum Taq reaction buffer and 4 µl of cDNA. The reaction mixture was preheated at 95??C for 5 minutes, followed by 45 cycles at 95??C for 15 seconds and 60??C for 1 minute. Amplification for COL-1, COL-3, MMP-2, MMP-8, MMP-13, MMP-14, TIMP-1, TIMP-2, and ß-glucuronidase could be demonstrated to be linear throughout a broad concentration range, allowing relative quantification in two independent runs using the CTC method as described.23,24 Briefly, the relative transcript level was expressed as the difference of the CTC values: CT . For example: CT = C3.5 means that this case expressed 9.5-fold more transcripts (1.9C(C3.5)) compared to the control group. For a comprehensive review of the equations underlying this quantification algorithm, see Livak and Schmittgen.23
Table 2. Primer and Probes Used Throughout This Study
DNA Extraction, JAK2 Amplification, and Restriction Site Analysis
The hotspot guanine-to-thymine point mutation in exon 12 of JAK2 destroys a relevant recognition site for the restriction enzyme BsaXI and avoids digestion of amplified JAK2 as described.5 We adopted this methodology for analysis of FFPE bone marrow cells.25 For DNA extraction from total bone marrow cells, one 10-µm slide was cut from the FFPE block before application of the DNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer??s instructions. PCR amplification of JAK2 was performed for 40 cycles in a GeneAmp PCR System 2700 (Applied Biosystems, Weiterstadt, Germany) using 2.5 mmol/L magnesium chloride and 25 ng of DNA. Primers for JAK2 amplification covering the hotspot mutation site corresponding to nucleotide 55.061 (GenBank sequence AL161450) were: JAK2 forward 5'-TATGATGAGCAAGCTTTCTCACAAG-3', and JAK2 reverse 5'-AGAAAGGCATTAGAAAGCCTGTAGTT-3' generating a 102-bp product.
Ten µl of a given sample were incubated with 4 U of the restriction enzyme BsaXI (2 U/µl; New England Biolabs, Beverly, MA) for 6 hours at 37??C in a PCR cycler with heated lid to avoid sample evaporation. The BsaXI enzyme recognized a site (5'... . 9(N) A C (N)5 C T C C (N)10... . 3'), which included the hotspot nucleotide 55.061 (GenBank sequence AL161450). For determination of BsaXI digestion in the respective JAK2 sequence, the NEBcutter 2.0 software tool was used (http://tools.neb.com/NEBcutter2/index.php). Samples analyzed by restriction site analysis were strictly accompanied by a positive control (JAK2 mutated cell line HEL) and a negative control (normal hematopoiesis).
JAK2 (V617F) Genotyping and Allele Quantification by Pyrosequencing
We established a pyrosequencing assay to quantify the percentage of mutant alleles in bone marrow cells from different disease stages of cIMF. The 102-bp fragment covering the hotspot point mutation (1849G>T) was amplified by PCR from 25 ng of genomic DNA with the primer combination used for BsaXI restriction site analysis with an additional 5'-biotin tag in a final volume of 50 µl. Forty-five µl of PCR product were mixed for 5 minutes (1200 rpm) at room temperature with 3.0 µl Streptavidin Sepharose HP (Amersham Biosciences, Freiburg, Germany) and 47 µl of binding buffer (Biotage, Uppsala, Sweden) using a thermomixer (Eppendorf, Hamburg, Germany). Using the Vacuum Prep tool (Biotage), single-stranded PCR products were prepared for sequencing analysis. Templates attached to the beads were washed with 70% ethanol for 5 seconds, denatured in 0.5 mol/L NaOH solution for 10 seconds, and washed in washing buffer (Biotage) for 5 seconds. Then the vacuum was switched off, and the beads were released into a PSQ 96 Plate Low (Biotage) containing 45 µl of annealing buffer (Biotage) and 500 nmol/L sequencing primer (5'-GGTTTTAAATTATGGAGTATGT-3', nucleotides 55.039 to 55.060 in GenBank AL161450).
The samples were heated to 80??C for 2 minutes and then cooled to room temperature. Pyrosequencing reactions were performed in a PSQ 96MA instrument (Biotage) according to the manufacturer??s instructions using the PyroGold SNP reagent kit (Biotage), which contains enzyme, substrate mixture, and nucleotides. Allele frequency was quantified using the SNP software (Biotage). As described, samples were scored as heterozygous for the JAK2 mutation if the percentage of mutant alleles exceeded 5%.26 Homozygosity was considered if the percentage of mutant T alleles exceeded 50%.4
Immunohistochemistry
To delineate cellular sources for MMP-14 (MT1-MMP), bone marrow showing cellular, prefibrotic cIMF (n = 10), advanced cIMF (n = 11), and control cases representing either normal hematopoiesis (n = 11) or reactive megakaryocytic hyperplasia (n = 10) were stained with a monoclonal anti-human MMP-14 antibody (MAB918; R&D Systems, Minneapolis, MN) recognizing the catalytic domain of the mature peptide. Bone marrow sections (2 µm) were stained with the Ready-to-use (Vector Laboratories). Vectastain ABC kit and the peroxidase DAB substrate kit (no. PK-7200 and no. SK-4100, respectively; R.T.U., Burlingame, CA) according to the manufacturer??s instructions. Briefly, sections were pretreated in citrate buffer, pH 6.0, in a microwave oven at 900 W and 750 W, 10 minutes each. Endogenous peroxidase was blocked by incubation with 3% H2O2 at room temperature for 10 minutes followed by incubation with normal horse serum for 1 hour at room temperature. Sections were incubated overnight with the primary anti-human MMP-14 antibody at 4??C. After overnight incubation the sections were incubated with the secondary antibody and ABC components for 30 minutes each. DAB staining was performed for 2 minutes at room temperature followed by counterstaining using hemalum according to standard protocols. FFPE colon carcinoma tissue (R&D Systems) served as the positive control according to the distributor??s instructions. Negative controls were performed for each staining procedure by omitting the secondary antibody.
Statistical Analysis
To analyze differences of gene expression in cellular prefibrotic cIMF, advanced cIMF, and nonneoplastic hematopoiesis, nonparametric Kruskal-Wallis tests were performed followed by Mann-Whitney U-tests for pair-wise group differences (P values Bonferroni-adjusted). Kruskal-Wallis tests were also performed to analyze differences of gene expression in V617F JAK2-mutated and wild-type JAK2 in both cellular cIMF and advanced cIMF. P values 0.05 were considered as statistically significant.
Results
Comparison of JAK2 Mutation Status and Expression of Genes Involved in the Turnover of Fibers
The overall frequency of V617F JAK2 mutations in the study group was 45% (9% homozygosity, 36% heterozygosity) in cellular cIMF and 53% (13% homozygosity, 40% heterozygosity) in advanced cIMF. Control cases under study showed entirely wild-type JAK2 (Table 3) . Heterozygous V617F JAK2, homozygous V617F JAK2, and wild-type JAK2, as evidenced by our pyrosequencing assay, are shown in Figure 1 . Restriction site analysis and pyrosequencing rendered identical results. The JAK2 mutation status or the wild-type state did not correlate with expression of MMPs, TIMPs, and COLs except for TIMP-2, which showed a higher level in cellular cIMF cases with wild-type JAK2 (P = 0.003). A comprehensive analysis of potential correlation of JAK2 status and gene expression is summarized in Table 4 .
Table 3. JAK2 Mutation Status in the Study Group
Figure 1. Genotyping analyses by the V617F JAK2 pyrosequencing assay are shown. The heights of peaks as shown in the pyrogram traces are proportional to the amount of nucleotide in the sequenced DNA. The mutation site is displayed in yellow and represents the potential hot spot and an adjacent normal T. Pyrosequencing of a case heterozygous for the V617F JAK2 mutation showed 60% G and 40% for the mutant T allele (A); a case homozygous for the mutation showed 13% G and 87% for the mutant T allele (B). A wild-type control case showed 100% G and no mutant T allele at all (C).
Table 4. JAK2 Mutation Status and Relation to Stage-Specific Expression Levels of MMPs, TIMPs, and COLs
Aberrant Expression of Collagenase Subsets (MMP-8, MMP-13, MMP-14/MT1-MMP) Define Different Stages of Disease in cIMF
MMP-14 mRNA was demonstrated to be significantly overexpressed in advanced cIMF (n = 33) by up to sevenfold (median, 2.4; range, 0.5 to 7.1) compared to prefibrotic cIMF (median, 0.9; range, 0.3 to 2.0; P < 0.001; n = 31) and control hematopoiesis (median, 0.8; range, 0.5 to 2.0; P < 0.001; n = 17) (Figure 2F) . MMP-13 mRNA levels showed differences between advanced cIMF (median, 4.6; range, 0.5 to 83.2; n = 32) and the cellular, prefibrotic phase (median, 0.7; range, 0.2 to 2.4; P < 0.001; n = 23). Normal hematopoiesis (median, 1.2; range, 0.2 to 4.3; n = 16) did not differ from either cIMF stage (Figure 2E) . MMP-8 mRNA was significantly up-regulated in cellular, prefibrotic cIMF (n = 25) by up to sixfold (median, 2.2; range, 0.3 to 6.0) as compared to control hematopoiesis (median, 1.0; range, 0.3 to 2.8; P < 0.001; n = 24). Cellular cIMF and advanced cIMF (median, 1.2; range, 0.5 to 11.5; n = 15) did not differ significantly from each other (Figure 2D) .
Figure 2. COL1, COL3, MMP-13, and MMP-14 showed significantly increased expression in advanced cIMF (A, B, E, and F, respectively). MMP-8 mRNA was exclusively up-regulated in prefibrotic cIMF compared to normal hematopoiesis but showed no differences to advanced cIMF (D). Apart from single outliers, no differences could be demonstrated in gene expression of MMP-2 and the inhibitors TIMP-1 and TIMP-2 (C, G, and H, respectively). Note that single exaggerated target gene expression levels were not included in the point plots (A: one case with advanced cIMF showing a 41-fold increase of COL1; B: two advanced cIMF cases with a 25.1-fold and 76.3-fold overexpression of COL3; C: two outliers with cellular cIMF showing 41.4-fold and 131.4-fold overexpression of MMP-2; E: four cases with advanced cIMF showing a 28.7-fold, 32.6-fold, 34.2-fold, and 83.2-fold increase of MMP-13 expression).
Significant Up-Regulation of Collagen Genes Defines the Advanced Stage in cIMF
COL-3 mRNA was significantly increased in advanced cIMF by up to 76-fold (median, 5.3; range, 1.0 to 76.3; n = 33) compared to cellular, prefibrotic cIMF (median, 1.2; range, 0.3 to 4.3; P < 0.001; n = 31) and normal hematopoiesis (median, 0.6; range, 0.01 to 3.3; P < 0.001; n = 24). The latter did not differ from each other (Figure 1B) . COL-1 gene expression in advanced cIMF (n = 33) was increased by up to 41-fold (median, 2.4; range, 0.4 to 41.0) as compared to the prefibrotic phase (median, 0.6; range, 0.1 to 9.8; P < 0.001; n = 31) and normal hematopoiesis (median, 1.2; range, 0.1 to 9.5; P = 0.03; n = 24) (Figure 2A) .
MMP-2, TIMP-1, and TIMP-2 mRNA Are Constitutively Expressed in cIMF and Normal Hematopoiesis
Prefibrotic cIMF (median, 1.7; range, 0.3 to 131.4; n = 21), advanced cIMF (median, 1.5; range, 0.1 to 7.8; n = 15), and control hematopoiesis (median, 1.3; range 0.3 to 5.7; n = 21) did not differ significantly from each other with regard to expression of MMP-2 mRNA (Figure 1C) . TIMP-1 mRNA was also constitutively expressed to similar levels by bone marrow cells in cellular, prefibrotic cIMF (median, 1.0; range, 0.2 to 5.3; n = 21), advanced cIMF (median, 1.9; range, 0.8 to 5; n = 15), and control hematopoiesis (median, 1.3; range, 0.1 to 3.0; n = 21) without notable differences (Figure 2G) . TIMP-2 mRNA in prefibrotic cIMF (median 1.1; range 0.3 to 2.3; n = 13), advanced cIMF (median, 1.0; range, 0.1 to 2.8; n = 14), and normal hematopoiesis (median, 1.0; range, 0.6 to 1.5; n = 10) also did not differ from each other (Figure 2H) . For a comprehensive illustration of the entire spectrum of gene expression level, see Table 4 .
Dynamics of MMP, TIMP, and COL mRNA Expression during the Course of Disease in Progressive Myelofibrosis and Stable Prefibrotic Stages
At presentation of case 1, histopathological evaluation of the bone marrow revealed megakaryocytic and granulocytic proliferation suspicious for a prefibrotic cIMF (mf 0). A control biopsy taken 5 months later revealed prefibrotic cIMF without notable fiber deposition (mf 0). Consecutive sequential trephine biopsies 1 year (mf 2) and 3 years later (mf 3) showed progressive myelofibrosis. The onset of demonstrable fiber deposition was paralleled by increased expression of MMP-14, MMP-13, and COL-3 genes. The manifest myelofibrosis 3 years after initial diagnosis exhibited decreased TIMP-1 and MMP-8 levels (Figure 3) .
Figure 3. In progressive myelofibrosis, MMP-14 mRNA expression notably increased at the onset of demonstrable fiber deposition along with COL3 and MMP-13. The stage of manifest fibrosis showed decreased TIMP-1 and MMP-8 levels whereas considerable fluctuations in mRNA expression were demonstrable for MMP-2 and MMP-13. Note that the expression level of a given target gene under investigation at initial diagnosis was set to 1. Representative silver impregnations (Gomori) of bone marrow sections revealed progressive myelofibrosis starting in June 2001. This course of disease showed wild-type JAK2 at any time point of investigation. Note that expression level in the first biopsy was set to 1.
In case 2 the prefibrotic stage persisted (Figure 4) and was reflected by constantly increased MMP-2 and increased MMP-13. In contrast to progressive myelofibrosis, no change in the expression of MMP-14 was demonstrable. A definite decline in the expression of COL-1 during the course of disease was paralleled by constant TIMP expression. Of note, no notable histopathological change was demonstrable in the sequential biopsies at the evaluation of time points. In these two exemplary courses of disease, no correlation between development of myelofibrosis and occurrence of JAK2 mutation could be demonstrated. The wild-type JAK2 case showed progressive myelofibrosis (Figure 3) whereas course 2 showed transition from the heterozygous V617F JAK2 status to homozygosity during the follow-up (Figure 4) .
Figure 4. The persistence of the prefibrotic stage in a disease course was reflected by persistently increasing MMP-2 and MMP-13 levels restricted to the early phase. In contrast to progressive myelofibrosis, no change in the expression of MT1-MMP was demonstrable. A definite decline in the expression of COL1 could be shown in the disease course paralleled by no change in TIMP expression. Silver impregnation (Gomori) of sequential bone marrow trephines revealed no notable deposition of either reticulin (COL3) or collagen fibers throughout this course of cellular, prefibrotic cIMF. An initial heterozygous state in the years 2000 and 2001 switched to homozygosity in October 2003. Note that expression level in the first biopsy was set to 1.
MMP-14 Protein Is Predominantly Localized in Endothelial Cells and Megakaryocytes
An intense staining could be observed in advanced stages of cIMF with stronger labeling of megakaryocytes, granulocytic precursors, and endothelial lining of sinusoids (Figure 5A) . In normal and reactive bone marrow, MMP-14 (MT1-MMP) immunohistochemistry revealed a weak and inconstant decoration of megakaryocytes (Figure 5B) .
Figure 5. Endothelial cells and megakaryocytes in advanced cIMF were prominently stained for MMP-14 (MT1-MMP) protein. A: Labeling of endothelial cells (arrows) was demonstrable in extended sinusoids (arrows) along with staining of clustered megakaryocytes and also immature granulopoiesis. B: Normal hematopoiesis showed heterogeneously labeled megakaryocytes and also occasionally granulocytic precursors. Original magnifications: x400 (A); x100 (B).
MMP-1 Is Rarely Detectable in Bone Marrow Cells Derived from Normal Hematopoiesis and cIMF
Two different primer systems for amplification of MMP-1 (Table 2) in bone marrow cells were tested. The overall detection rate in a qualitative RT-PCR assay was 30% in both cIMF (n = 12) and normal hematopoiesis (n = 12). MMP-1 RT-PCR amplicons derived from cIMF and normal hematopoiesis were almost undetectable as demonstrated by comparison with MMP-1 amplicons derived from a fibroblast cell line (positive control; data not shown).
Discussion
Until now investigations on the role of MMPs and collagen deposition in cIMF have been hampered due to punctio sicca (dry tap) particularly in advanced stages. An imbalance of increased collagen synthesis and decreased proteolytic action in cIMF seems to be a plausible underlying mechanism. Previous studies investigated plasma of patients with cIMF and related Philadelphia chromosome-negative chronic myeloproliferative disorders for aberrant MMP and TIMP protein levels and suggested that elevated TIMP together with decreased MMP levels might be essential for fibrosis formation.27,28 Data on serum markers of collagen metabolism and expression of members of the urokinase-type plasminogen activator system (uPA) suggested an impact on remodeling processes during myelofibrosis.29 A more recent study investigated potential mechanisms of enhanced CD34+ cell trafficking in cIMF and demonstrated significantly increased plasma and cellular levels of neutrophil elastase (NE) and MMP-9 along with soluble vascular adhesion molecule-1 (sVCAM-1). However, mononuclear cells from cIMF patients in this study served as a surrogate because bone marrow cells could not be harvested because of manifest myelofibrosis.14
We took advantage of recent progress in molecular analysis of FFPE trephine biopsies30 and investigated total bone marrow cells of prefibrotic and advanced cIMF for expression of collagens and a broad spectrum of MMPs that preferably degrade collagens. Because the JAK-STAT pathway has been demonstrated to be involved in the regulation of MMP genes and TIMPs,15-17 we further tested the hypothesis that cIMF cases showing the V617F JAK2 mutation might differ from wild-type JAK2 cases.
In addition to this, expression of the entire spectrum of MMPs and COLs under investigation showed no correlation to the absence or evidence of an underlying V617F JAK2 mutation in a given case. In particular, the state of zygosity in V617F JAK2 mutated cases in both cIMF groups had no effect on target gene expression. TIMP-2, but not TIMP-1, showed a rather higher expression in cellular cIMF with wild-type JAK2 compared to V617F JAK2 in this group but not in comparison to advanced cIMF and control cases. Not unexpectedly, COL-1 and -3 were increased in advanced cIMF, reflecting the ongoing process of collagen synthesis at this stage. Interestingly, in both prefibrotic and advanced cIMF, no MMP examined here showed a decreased expression compared to normal hematopoiesis. Most notably, MMP-14 and MMP-13 could be demonstrated to be significantly increased in advanced cIMF stages. MMP-14 exhibits various important properties including direct cleavage of collagen fibers, activation of other MMPs (such as MMP-13), and induction of angiogenesis.31 In contrast to previous studies that propagated the interaction of multiple MMPs such as MMP-2/MMP-9 and integrins for sufficient angiogenesis,32,33 more recent data have demonstrated the essential role for MMP-14 in this process.34 Tissues from MMP-14 knockout mice failed to develop vessels in collagen matrices whereas tissues from mice knocked out for MMP-2, MMP-9, and CD44 generated normal vessels. Increased angiogenesis is a hallmark in the bone marrow architecture of advanced cIMF as demonstrated by increased microvessel density or labeling of endothelial osteoprotegerin.35,36 Besides megakaryocytes and myeloid precursor cells, we probably unmasked endothelial cells in advanced cIMF as a major source for MMP-14, suggesting its impact on proliferating vessels in the disease course (Figure 5) . Because hematopoietic and endothelial progenitor cells are regularly increased in the peripheral blood of patients with cIMF,37 MMP-14 might play a central role in abnormal cell trafficking. This hypothesis is supported by the conclusion of a previous study that a cell-bound MMP could be involved in increased mobilization of cIMF progenitors.14 Moreover, a recent study revealed MMP-14 as the key pericellular collagenolysin in the process of pathological vessel remodeling and angiogenesis.38
The investigation of COLs and MMPs in both disease stages of cIMF represents only a snap-shot in the dynamic process of matrix remodeling. To gain more insight into the intraindividual expression, we selected two disease courses showing either progressive myelofibrosis or persistence of the prefibrotic stage in a comparable time frame. Clear differences in the expression of MMPs (MMP-14, MMP-13, MMP-2) and both COLs were demonstrable between the two courses. Different dynamics in gene expression and the missing correlation of JAK2 status and fibrosis were evident, but investigation of a larger series of sequential biopsies in the course of cIMF is needed to prove reproducibility. Accordingly, these figures per se do not allow us to decide on a course in a prospective manner. In general, it is difficult to illustrate the dynamics of ECM remodeling appropriately because activation of latent MMPs is required but not sufficiently demonstrable, neither by gene expression nor by immunohistochemistry. However, as demonstrated not only for single-case analysis but also in the two courses, the increased MMP-14 levels in progressive myelofibrosis shed light on a probably important candidate in this process.
There is no doubt that MMPs and TIMPs represent only two important systems14,27-29 in the complex network of proteolytic enzymes and specific inhibitors. Other factors such as the uPA/plasminogen/plasmin/PAI system, elastases, or stromelysins concomitantly interact in the process of ECM remodeling in cIMF. In addition, deposition of matrix components such as collagens in cIMF is boosted by cytokine action such as interleukins thereby further complicating the network of aberrant mechanisms.
Of note, the presence of the JAK2 (V617F) mutation in all cases under investigation did not correlate with a case-specific fibrogenic potential. In a larger series of sequential biopsies in cIMF not yet investigated for targets involved in ECM remodeling, progressive myelofibrosis was also demonstrable in JAK2 wild-type cases (such as no. 1, Figure 3 ) or longer lasting prefibrotic stages (such as no. 2, Figure 4 ) showing JAK2 mutation at initial diagnosis (not published). Therefore, constitutive catalytic activity mediated by mutated JAK2 does not seem to be required for any fibrogenic signals.
We conclude that ECM remodeling in disease stages of cIMF is reflected by aberrant expression of defined collagenase subsets independent of an underlying V617F JAK2 mutation.
Acknowledgements
We thank Ms. Christina Koop and Ms. Sabine Schroeter for their skilled technical assistance; and Dr. Masyar Monazahian, Niedersächsisches Landesgesundheitsamt, Hannover, Germany, for the opportunity to perform the pyrosequencing assay on the PSQ 96MA instrument.
【参考文献】
Brunning RD, Flandrin G, Imbert M, Pierre R, Thiele J, Vardiman JW: Chronic myeloproliferative diseases. Jaffe ES Harris NL Stein H Vardiman JW eds. WHO classification of tumours. Pathology and Genetics. Tumours of Haematopoietic and Lymphoid Tissues. 2001:pp 15-42 IARC Press Lyon
Tefferi A: Pathogenesis of myelofibrosis with myeloid metaplasia. J Clin Oncol 2005, 23:8520-8530
Kaushansky K: On the molecular origins of the chronic myeloproliferative disorders: it all makes sense. Blood 2005, 105:4187-4190
Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC: A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005, 352:1779-1790
Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR: Cancer Genome Project. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005, 365:1054-1061
James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, Garcon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W: A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005, 434:1144-1148
Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D??Andrea A, Frohling S, Dohner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG: Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005, 7:387-397
Stamenkovic I: Extracellular matrix remodelling: the role of matrix metalloproteinases. J Pathol 2003, 200:448-464
Gupta P, Oegema TR, Jr, Brazil JJ, Dudek AZ, Slungaard A, Verfaillie CM: Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche. Blood 1998, 92:4641-4651
Chichester CO, Fernandez M, Minguell JJ: Extracellular matrix gene expression by human bone marrow stroma and by marrow fibroblasts. Cell Adhes Commun 1993, 1:93-99
Buhr T, B?sche G, Choritz H, Länger F, Kreipe H: Evolution of myelofibrosis in chronic idiopathic myelofibrosis as evidenced in sequential bone marrow biopsy specimens. Am J Clin Pathol 2003, 119:152-158
Thiele J, Kvasnicka HM: Prefibrotic chronic idiopathic myelofibrosis??a diagnostic enigma? Acta Haematol 2004, 111:155-159
Visse R, Nagase H: Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003, 92:827-839
Xu M, Bruno E, Chao J, Huang S, Finazzi G, Fruchtman SM, Popat U, Prchal JT, Barosi G, Hoffman R: MPD Research Consortium. Constitutive mobilization of CD34+ cells into the peripheral blood in idiopathic myelofibrosis may be due to the action of a number of proteases. Blood 2005, 105:4508-4515
Ratajczak J, Majka M, Kijowski J, Baj M, Pan ZK, Marquez LA, Janowska-Wieczorek A, Ratajczak MZ: Biological significance of MAPK, AKT and JAK-STAT protein activation by various erythropoietic factors in normal human early erythroid cells. Br J Haematol 2001, 115:195-204
Li WQ, Dehnade F, Zafarullah M: Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway. J Immunol 2001, 166:3491-3498
Bartolome RA, Molina-Ortiz I, Samaniego R, Sanchez-Mateos P, Bustelo XR, Teixido J: Activation of Vav/Rho GTPase signaling by CXCL12 controls membrane-type matrix metalloproteinase-dependent melanoma cell invasion. Cancer Res 2006, 66:248-258
Nguyen J, Knapnougel P, Lesavre P, Bauvois B: Inhibition of matrix metalloproteinase-9 by interferons and TGF-beta1 through distinct signalings accounts for reduced monocyte invasiveness. FEBS Lett 2005, 579:5487-5493
Andl CD, Mizushima T, Oyama K, Bowser M, Nakagawa H, Rustgi AK: EGFR-induced cell migration is mediated predominantly by the JAK-STAT pathway in primary esophageal keratinocytes. Am J Physiol 2004, 287:G1227-G1237
Ribatti D, Presta M, Vacca A, Ria R, Giuliani R, Dell??Era P, Nico B, Roncali L, Dammacco F: Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo. Blood 1999, 93:2627-2636
Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der Walt J, Orazi A: European consensus on grading bone marrow fibrosis and assessment of cellularity. Haematologica 2005, 90:1128-1132
Bock O, Kreipe H, Lehmann U: One-step extraction of RNA from archival biopsies. Anal Biochem 2001, 295:116-117
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method. Methods 2001, 25:402-408
Bock O, Loch G, Schade U, von Wasielewski R, Schlue J, Kreipe H: Aberrant expression of transforming growth factor beta-1 (TGF beta-1) per se does not discriminate fibrotic from non-fibrotic chronic myeloproliferative disorders. J Pathol 2005, 205:548-557
Bock O, Buesche G, Koop C, Schroeter S, Buhr T, Kreipe H: Detection of the single hotspot mutation in the pseudokinase domain JH2 of Janus kinase 2 (JAK2) in bone marrow trephine biopsies from chronic myeloproliferative disorders. J Mol Diagn 2006, 8:170-177
Jelinek J, Oki Y, Gharibyan V, Bueso-Ramos C, Prchal JT, Verstovsek S, Beran M, Estey E, Kantarjian HM, Issa JP: JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML, Philadelphia chromosome-negative CML, and megakaryocytic leukemia. Blood 2005, 106:3370-3373
Wang JC, Novetsky A, Chen C, Novetsky AD: Plasma matrix metalloproteinase and tissue inhibitor of metalloproteinase in patients with agnogenic myeloid metaplasia or idiopathic primary myelofibrosis. Br J Haematol 2002, 119:709-712
Wang JC: Importance of plasma matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinase (TIMP) in development of fibrosis in agnogenic myeloid metaplasia. Leuk Lymphoma 2005, 46:1261-1268
Jensen MK, Riisbro R, Holten-Andersen MN, Brown Pde N, Junker P, Brunner N, Hasselbalch HC: Collagen metabolism and enzymes of the urokinase plasminogen activator system in chronic myeloproliferative disorders: correlation between plasma-soluble urokinase plasminogen activator receptor and serum markers for collagen metabolism. Eur J Haematol 2003, 71:276-282
Lehmann U, Kreipe H: Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 2001, 25:409-418
Itoh Y, Seiki M: MT1-MMP: a potent modifier of pericellular microenvironment. J Cell Physiol 2006, 206:1-8
Brooks PC, Silletti S, von Schalscha TL, Friedlander M, Cheresh DA: Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity. Cell 1998, 92:391-400
Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D: Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2000, 2:737-744
Chun TH, Sabeh F, Ota I, Murphy H, McDonagh KT, Holmbeck K, Birkedal-Hansen H, Allen ED, Weiss SJ: MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix. J Cell Biol 2004, 167:757-767
Mesa RA, Hanson CA, Rajkumar SV, Schroeder G, Tefferi A: Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia. Blood 2000, 96:3374-3380
Bock O, Loch G, Schade U, Buesche G, Wasielewski R, Wiese B, Kreipe H: Osteosclerosis in advanced chronic idiopathic myelofibrosis is associated with endothelial overexpression of osteoprotegerin. Br J Haematol 2005, 130:76-82
Barosi G, Hoffman R: Idiopathic myelofibrosis. Semin Hematol 2005, 42:248-258
Filippov S, Koenig GC, Chun TH, Hotary KB, Ota I, Bugge TH, Roberts JD, Fay WP, Birkedal-Hansen H, Holmbeck K, Sabeh F, Allen ED, Weiss SJ: MT1-matrix metalloproteinase directs arterial wall invasion and neointima formation by vascular smooth muscle cells. J Exp Med 2005, 202:663-671
作者单位：From the Institutes of Pathology* and Biometrics, Hannover Medical School, Hannover, Germany