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发表于 2009-3-4 23:57 |只看该作者 |倒序浏览 |打印

作者：Roberto M. Lemolia,b, Lucia Catania,b, Simona Talaricoa,b, Elisabetta Loggia,c, Annagiulia Gramenzia,c, Umberto Baccaranid, Miriam Foglia,b, Gian Luca Grazie, Michela Aluigia,b, Giulia Marzocchia,b, Mauro Bernardia,c, Antonio Pinnae, Fabrizio Bresadolad, Michele Baccarania,b, Pietro Andreonea,c作者单位：aInstitute of Hematology and Medical Oncology L. A. Sergnoli, Bologna, Italy;


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      【摘要】& ?8 U. b; C4 M
   In animals, the bone marrow (BM) is a source of liver-repopulating cells with therapeutic potential in case of tissue damage. However, the early response of human BM-derived stem cells (SC) to liver injury is still unknown. Here, we studied 24 patients undergoing orthotopic liver transplantation (OLT) for end-stage liver disease or hepatocellularcarcinoma, and 13 patients submitted to liver resection. The concentration of circulating BM-derived SC was determined by phenotypic analysis and clonogenic assays. Moreover, we assessed the serum level of inflammatory and tissue-specific cytokines. Reverse transcriptase-polymerase chain reaction and fluorescence-in situ hybridization were also used to characterize mobilized SC. At baseline, patients showed a significant lower concentration of circulating CD133 , CD34  SC and clonogenic progenitors (colony-forming unit cells) than healthy controls. However, the time-course evaluation of peripheral blood cells after OLT demonstrated the significant early mobilization of multiple subsets of hematopoietic and endothelial stem/progenitor cells. Cytogenetic and molecular analyses of CD34  cells showed the host origin of mobilized SC and the expression of transcripts for GATA-4, cytokeratin 19, and -fetoprotein hepatocyte markers. In contrast with OLT, only total circulating CD34  cells significantly increased after liver resection. Mobilization of BM cells after OLT or liver surgery was associated with increased serum levels of granulocyte-colony stimulating factor, interleukin-6, stem cell factor, hepatocyte growth factor, and vascular endothelial growth factor. In summary, we demonstrate that tissue damage after OLT and liver resection induces increased serum levels of multiple cytokines but only ischemia/reperfusion injury associated with OLT results in the remarkable mobilization of BM stem/progenitor cells.
      【关键词】 Orthotopic liver transplantation Spontaneous mobilization Hematopoietic and endothelial stem cells
   INTRODUCTION9 l- r( d5 A# `% Y, Q8 b

At least two resident cell populations have been shown to act as putative stem cells (SC) in the liver .
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Recently, a third source of liver-repopulating cells has proven to be the bone marrow (BM) as animal studies have shown that BM cells contribute, at low levels, to liver regeneration after tissue injury .
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The potential of human CD34  HSCs to migrate to the liver in response to stress signals to repair non-hematopoietic tissue has been recently established in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice . In both experimental models, G-CSF administration improved the histological damage and accelerated the regeneration process. These findings translated into a strong survival benefit in G-CSF-treated group versus CCl4 group. Thus, G-CSF treatment significantly improved the liver histology of chemically injured mice by promoting endogenous repair mechanisms and by mobilizing HSCs.

Despite extensive investigations in animal models of liver injury . In this investigation, we studied the early response of BM-derived SC of patients undergoing orthotopic liver transplantation (OLT) or liver resection.
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Our phenotypic, functional and molecular studies demonstrated: (a) the significant, physiologic mobilization of host-derived hematopoietic and endothelial stem/progenitor cells after tissue injury; (b) this finding was associated with increased serum levels of cytokines involved in SC mobilization and/or liver repair; (c) ischemia/reperfusion organ damage associated with OLT is a more efficient stimulus to SC mobilization than massive liver resection.: v' m8 K& h& s* v6 _3 w: o& J5 x7 D

MATERIALS AND METHODS9 b& }& }( |1 F
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Patients and Control Group! n( V- v# W1 L/ B& A
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We assessed the peripheral blood (PB) stem/progenitor cells compartment of 24 and 13 adult patients undergoing OLT and liver resection, respectively. The clinical characteristics of study patients are reported in Table 1. Blood samples were taken one day before (day ¨C1) and then one, three, seven, and 14 days after surgery when possible. Single samples from 12 healthy subjects served as controls. Study patients underwent liver surgery according to standard procedures either at the Liver and Multi Organ Transplant Center of the University of Bologna, Bologna, Italy or at the Department of Surgery and Organ Transplantation, University of Udine, Udine, Italy. The study protocol was approved by the local Ethic Committees and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained by all patients and healthy controls.) e: D/ C, S) `9 Y

Table 1. Characteristics of patients
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Flow Cytometry Analysis' C! o8 n. t( u" ~$ }

The phenotype of circulating cells was evaluated by conventional dual color immunofluorescence using a panel of fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated monoclonal antibodies (MoAbs): FITC-conjugated anti-CD34, PE-conjugated anti-CD38, PE-conjugated anti-CXCR4, PE-conjugated anti-CD90 (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). PE-conjugated anti-CD133 was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). MoAbs against vascular endothelial growth factor receptor (VEGFR)-2 (KDR) (clone KDR-1), VEGFR-1 (Flt-1 receptor) (clone FLT-19) (Sigma, Saint Louis, MI, http://www/sigmaaldrich.com) antigens were tested by indirect immunofluorescence. Negative controls were isotype-matched irrelevant MoAbs (Becton Dickinson). Briefly, 100 µl of PB in heparin-containing tubes (Becton Dickinson) were incubated for 15 minutes at room temperature with 10 µl of MoAbs. In case of indirect immunofluorescence, cells were then incubated for 15 minutes with 5 µl of PE/FITC-conjugated goat anti-mouse immunoglobulins (Becton Dickinson). After red-cell lysis (FACS Lysing Solution, Becton Dickinson), the samples were centrifuged, washed twice with phosphate buffer and fixed with 1% paraformaldehyde (Sigma). Five x 104 cells were acquired by flow cytometer (FACSCalibur; Becton Dickinson) and analyzed by CellQuest software (Becton Dickinson) . The analysis was performed excluding cellular debris in a side scatter/forward scatter dot plot. The percentage of positive cells was calculated subtracting the value of the appropriate isotype controls. The absolute number of positive cells per microliter was calculated as follows: percentage of positive cells x white blood cells count/100.
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Hematopoietic Colony-Forming Unit Cells Assays! S- l# `0 X* k: G8 v) H! j
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Assessment of clonogenic hematopoietic progenitors was performed in methylcellulose as previously reported .

Cell Preparation and HSC Purification" j  n' C$ a6 z

Enriched MNC were resuspended in 1% bovine serum albumin (BSA) (Sigma) and then processed by MiniMacs high-gradient magnetic separation column (Miltenyi Biotec) to obtain highly purified CD34  cells as already reported . To assess the percentage of CD34  elements, aliquots of the CD34  target cells were restained with a MoAb (HPCA-2-FITC; Becton Dickinson) directed to a different epitope of CD34 antigen than that (Qbend 10) used with the magnetic system. Propidium iodide (2 µg/ml) was added for the detection of nonviable cells, which were excluded from the analyses. Cytometric analysis was performed on a gated population set on scatter properties by using FACScan equipment (Becton Dickinson). A minimum of 10,000 events was collected in list mode on FACScan software. The percentage of CD34  cells after magnetic separation was 93 ¡À 6%.* U) j1 ]" P1 x

Fluorescence In Situ Hybridization and Reverse Transcriptase-Polymerase Chain Reaction9 @9 Q+ ^5 e/ |3 Q) [2 `: P1 D

Both fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were performed on immunomagnetically highly purified CD34  cells. The slides were prepared immediately before hybridization. We used two probes: CEP X that hybridizes to centromere region Xp11.1-q11.1 (Spectrum Orange, Vysis Inc., Downers Grove, IL, http://www.vysis.com) and CEP Y to chromosome band Yq13 (satellite III, Spectrum Green, Vysis Inc.). The normal female pattern was two red signals, and the normal male pattern was one red and one green signal. Hybridization was performed overnight at 37¡ãC on a Hybrite semiautomated FISH hybridization chamber (Vysis), according to the manufacturer's guide with some modifications. Slides were mounted and counterstained with 4',6-diamidino-2-phenylindole (DAPI) in antifade. The samples were examined with a Nikon E-1000 fluorescent microscope equipped with FITC/tetramethylrhodamine isothiocyanate/DAPI filter and a Genikon FISH system image capture (Nikon Instruments, Rockville, MD, http://www.nikonusa.com). Two-hundred and ten CD34  HSC were analyzed.

The expression of GATA-4, cytokeratin (CK)-19 and -fetoprotein (AFP) transcripts was assessed by RT-PCR amplification using gene-specific primers (GATA-4, F: 5'-aggcattacatacaggctcacc-3', R: 5'-ctgtggcctctatcacaagatg-3'; CK-19, F: 5'-atggccgagcagaaccggaa-3', R: 5'-ccatgagccgtcggtactcc-3'; AFP, F: 5'-tgcagccaaagtgaagagggaaga-3', R: 5'-catagcgagcagcccaaagaagaa-3') . In brief, total cellular RNA was extracted from PB mononuclear cells by TRIzol reagent (Invitrogen, Milan, Italy, http://www.invitrogen.com). Reverse transcription was performed using the SuperScript II First-Strand Synthesis System (Invitrogen) following manufacturer's instructions. The expression of different transcripts was assessed by PCR amplification following standard protocols. PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. RNA integrity was evaluated by the expression of ß2-microglobulin.

Cytokine Measurements; Q4 p6 g3 M' f$ ?

The serum levels of study cytokines was measured before OLT or liver resection and on days 1, 7, and 14 after surgery by high-sensitivity enzyme-linked immunosorbent assays. SCF, G-CSF, HGF, SDF-1, vascular endothelial growth factor (VEGF), and tumor necrosis factor- (TNF-) kits were from R&D Systems (Wiesbaden, Germany, http://www.rndsystems.com). Interleukin (IL)-6 kit was from Biosource International (Camarillo, CA, http://www.biosource.com).
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Statistical Analysis
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The results were expressed as median (range). The data were analyzed by the Wilcoxon test for paired data, Mann-Whitney test and by the Fisher's exact test. Spearman rank correlation test was used for correlation analysis. A p value ) V8 Z! a, U! N& }
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RESULTS* r, Y) t7 E0 v# D" \9 v
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OLT Patients Mobilize Hematopoietic and Endothelial Stem/Progenitor Cells1 }/ z8 g0 u# b$ u& l2 Y

At baseline, patients with liver disease showed decreased hematopoietic function as demonstrated by the significant lower concentration of PB CD133  (median 0.35 cells per microliter; range 0¨C1.8), CD34  (0 cells per microliter; 0¨C0.7) SC, and CFU-C (19 cells per microliter; 0¨C525) than in healthy donors, respectively (1.44 cells per microliter; 0¨C10.8), (0 cells per microliter; 0¨C1.6), (554.5 cells per microliter; 362.5¨C1100) (Fig. 1).

Figure 1. Impaired bone marrow (BM) function in patients with liver disease. Circulating BM-derived cells were evaluated by phenotypic analysis and clonogenic assay. Baseline concentrations of CD34 , CD133 cells, and CFU-C were significantly lower in patients undergoing liver resection and OLT, respectively, than in healthy controls. The bold line indicates the median value. Abbreviations: CFU-C, colony-forming unit cells; OLT, orthotopic liver transplantation.

Nonetheless, the longitudinal phenotypic evaluation of circulating cells after OLT demonstrated the small, but significant, early mobilization of CD34  HSCs (day  7 0.13 cells per microliter; 0¨C9.3; p = .014) (Fig. 2A), which remained higher than the baseline value until day  14 (0 cells per microliter; 0¨C12.1; p = .041) and returned to pretreatment levels on day  30 after OLT (data not shown). In keeping with the spontaneous mobilization of CD34  cells, we observed a significantly higher number of circulating CFU-C (85.6 CFU-C per milliliter; 0¨C1830; p = .028) on day  14 after transplantation (Fig. 2A). The number of CFU-C was higher than the baseline value when the results were expressed both as number of colonies/106 MNC and colonies/PB milliliter. Thus, we ruled out any confounding effect due to the possible increase of the white blood cells count after OLT.

Figure 2. Phenotypic and functional analysis of peripheral blood (PB) cells before and after orthotopic liver transplantation (OLT) demonstrates the mobilization of bone marrow-derived stem/progenitor cells. (A): Following OLT, we observed the significant early increase of hematopoietic and endothelial stem/progenitor cells and the mobilization of CFU-C on day  14. The bold line indicates the median value. (B): OLT induces the mobilization of host-derived hematopoietic stem cells coexpressing mRNA for liver-specific epithelial markers. Fluorescence-in situ-hybridization analysis was performed on purified circulating CD34  cells from a male recipient of a female-derived liver and no female cells (XX, orange/orange pattern) were observed. (C). CK-19 (i),AFP (ii), GATA-4 (iii) transcripts were assessed in circulating CD34  cells by reverse transcription-polymerase chain reaction. Lanes 1 and 2, PB mononuclear cells from mobilized healthy donors; lanes 3 and 5, baseline samples from two nonmobilizer OLT patients; lanes 4 and 6, day  7 post-OLT samples from the same nonmobilizers; lanes 7 and 9, baseline samples from two mobilizers OLT patients); lanes 8 and 10, day  7 post-OLT samples from the same mobilizers; lane 11, positive control (human liver); lane 12, negative control (no cDNA); lane 13, molecular weight markers. Abbreviations: AFP, -fetoprotein; ß2-M, ß2-microglobulin; CFU-C, colony-forming unit cells; CK, cytokeratin.
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When we analyzed selected subsets of the CD34  HSC population, we found a significant increase of very immature CD34 /CD90  cells (day  3 0 cells per microliter; 0¨C7.4 vs. day ¨C1 0 cells per microliter; 0¨C0.4; p = .043) and CD34  cells coexpressing the chemokine SDF-1 receptor CXCR4 (day  3 0.65 cells per microliter; 0¨C3.9, day  7 0.5 cells per microliter; 0¨C4.6, day  14 0.83 cells per microliter; 0¨C3 as compared to day ¨C1 0 cells per microliter; 0¨C0.7; p

Also the concentration of multipotent PB CD133  stem/progenitor cells and endothelial CD34 /VEGFR-2 (KDR)  progenitors (Fig. 2A) augmented following OLT reaching the statistical significance (p 0 x4 K$ d6 E* X

Interestingly, the cold ischemia time of the liver transplant (Table 1) directly correlated with the number of circulating CD133  cells at day  3 after OLT (r = .5; p = .01). No difference was found in terms of mobilization taking into account the etiology of liver disease, the immunosuppression regimen or the post-transplant complications such as acute allograft rejection.

Mobilized CD34  SC Are of Host Origin and Coexpress mRNAs for Epithelial Liver-Specific Markers
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In a representative case of sex-mismatch transplant, circulating CD34  cells from a male recipient of a female-derived liver, were purified and analyzed by FISH (Fig. 2B). Two-hundred and ten cells were analyzed, and no female cells (XX, orange/orange pattern) were observed. This result demonstrated the host origin of circulating CD34  cells and ruled out that the observed increase of PB HSCs may be due to hematopoietic cells carried over by the transplanted liver .( [8 x3 u: q+ [& W7 V$ `
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Moreover, the RT-PCR assay showed that circulating CD34  cells analyzed after OLT, but not baseline steady-state cells from the same patients or PB samples from G-CSF-mobilized healthy donors, expressed mRNA for GATA-4, CK-19, and AFP liver markers (Fig. 2C). Therefore, we demonstrated that OLT induces the release from the BM of HSCs expressing liver markers.

SC Mobilization in Patients Undergoing Liver Resection* L( [) g$ I: {$ V) O

Thirteen patients, mostly with primary (seven cases) or metastatic liver cancer (three cases) (Table 1), who were submitted to liver resection were also studied to answer the question of whether the surgical procedure, per se, induces SC mobilization and whether the removal of a large part of the liver (up to 55%) represents an efficient stimulus to recruit BM cells into PB.
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Similarly to OLT patients, resected individuals showed impaired BM function as demonstrated by the significantly reduced number of circulating CD34  (0 cells per microliter; 0¨C0), CD133  cells (0 cells per microliter; 0¨C1.36) and CFU-C (70 CFU-C per milliliter; 0¨C684) at baseline (Fig. 1). The longitudinal study performed after surgery showed only the significant mobilization (p = .028) of total CD34  cells (0 cells per micrliter; 0¨C1.2) at day  3 after surgery (Fig. 3). The extent and the length of mobilization of CD34  cells after liver resection was markedly lower than after OLT. PB endothelial stem/progenitor cells as well as selected subsets of HSCs did not increase in response to liver resection even after removal of 55% of the organ (data not shown).

Figure 3. Liver resection induces the mobilization of CD34  HSCs. The phenotype of PB cells of resected patients was evaluated by conventional immunofluorescence. Following liver resection we observed the early mobilization of total CD34  at day  3. The bold line indicates the median value.
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SC Mobilization Is Associated with Increased Serum Levels of Selected Cytokines
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When we analyzed the serum level of cytokines involved in SC mobilization and/or liver repair, we found that the baseline levels of IL-6 (60 pg/ml; 0¨C453) and HGF (1,866 pg/ml; 997¨C3,960) were significantly higher (p

Figure 4. Baseline serum levels of study cytokines. The baseline levels of IL-6 and HGF were significantly higher, and VEGF levels lower, in patients undergoing OLT than in healthy controls. In liver resected patients, HGF and SCF serum concentrations were significantly higher and lower, respectively, than in control samples. The bold line indicates the median value. Abbreviations: HGF, hepatocyte growth factor; IL, interleukin; OLT, orthotopic liver transplantation; SCF, stem cell factor; VEGF, vascular endothelial growth factor.0 S, w% s4 H; |! s  i3 K
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Figure 5. Orthotopic liver transplantation induces increased serum levels of multiple cytokines. After liver transplantation, we observed the significant increase of IL-6, SCF, VEGF and G-CSF but not of HGF. The serum level of SDF-1 significantly decreased at days  1 and  7 after transplantation. The bold line indicates the median value. Abbreviations: G-CSF, granulocyte colony-stimulating factor; HGF, hepatocyte growth factor; IL, interleukin; SCF, stem cell factor; SDF, stromal cell-derived factor; VEGF, vascular endothelial growth factor.

When serum cytokine levels were analyzed in patients undergoing liver resection, we found the significant increase of SCF, IL-6, VEGF, HGF, and G-CSF (Fig. 6). Of note, the serum level of SCF was remarkably higher after OLT (day  7 1,303 pg/ml; 682¨C1,867) than after liver resection (day  7 499 pg/ml; 313¨C913). Again, G-CSF peaked at day  1 and rapidly decreased to pretreatment levels.
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Figure 6. Liver resection augments the serum level of multiple cytokines. When serum cytokine levels were analyzed in patients undergoing liver resection, we found a significant increase of IL-6, HGF, SCF, VEGF, and G-CSF. The bold line indicates the median value. Abbreviations: G-CSF, granulocyte colony-stimulating factor; HGF, hepatocyte growth factor; IL, interleukin; SCF, stem cell factor; SDF, stromal cell-derived factor; VEGF, vascular endothelial growth factor.
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DISCUSSION1 h9 P* S: Y) j% F
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To the best of our knowledge, this is the first study reporting the mobilization of both hematopoietic and endothelial stem/progenitor cells after OLT. We thus provide a strong rationale for the clinical observation of human liver cells deriving from circulating cells of extrahepatic origin upon tissue injury . In the present study, we demonstrated that despite baseline impaired hematopoietic function patients undergoing OLT and ischemia/reperfusion damage have a 3.4-fold increase of circulating CD34  cells, which is appreciable within 24 hours of surgery, peaks at days  7 and  14, and normalizes within 30 days. Phenotypic and functional analyses further showed that both primitive CD34 /CD90  HSCs and more mature, committed CFU-C were mobilized into PB within 14 days of OLT. Of note, the early and significant elevation of the concentration of different subsets of hematopoietic and endothelial stem/progenitor cells into PB occurred despite similar values of circulating leukocytes before and after OLT.# c' x# t5 N4 v7 V+ Q

Mobilization of HSCs was likely enhanced by increased serum levels of IL-6, SCF, and G-CSF after OLT. In particular, G-CSF and SCF have been shown to enhance, in animals, the expression of the chemokine SDF-1 and its cognate receptor, CXCR4, on HSCs to recruit marrow cells to the injured tissue .

Similarly, mobilization of endothelial progenitors may be due to the increased serum level of VEGF following the vascular damage associated with OLT. As a matter of fact, circulating CD133  and CD34 /VEGFR-2  (KDR ) cells are known to contribute to neoangiogenesis after tissue ischemia and organ regeneration in animal models .) \- F0 z' k  e8 e+ U9 [
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Thus, it is conceivable that hematopoietic and endothelial progenitors can play different roles, if any, in tissue repair. Since regeneration of the injured organ involves the proliferation of parenchymal cells as well as neovascularization, it may well be that tissue injury also induces the activation of progenitors for endothelium. These findings support the concept that "plasticity" may not be restricted to a unique SC population but, rather, may be a general property of marrow cells that redirect their transcriptional program under appropriate stimuli. Of note, mobilization of CD133  hematopoietic and endothelial stem/progenitor cells directly correlated with the length of the cold ischemia of the transplanted organ. Thus, it may be hypothesized that the greater the extent of the tissue damage (reflected by the cold ischemia time) is, the higher SC mobilization is.
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FISH analysis formally demonstrated the host origin of mobilized BM-derived CD34  HSCs after OLT. In addition, molecular studies showed that only HSCs circulating after OLT, but not CD34  cells evaluated before surgery or purified from G-CSF-mobilized healthy donors, expressed mRNAs for liver-specific epithelial markers such as AFP and CK-19 and for the GATA-4 transcription factor. Therefore, our data support the hypothesis of the pool of tissue-committed BM SC readily available after tissue damage as previously proposed .4 \8 u; S1 u9 b0 k/ ~8 P
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Whereas ischemia/reperfusion liver damage associated with OLT induced the extensive mobilization of several subsets of hematopoietic and endothelial BM-derived SC, liver resection was a weaker stimulus to recruit significant numbers of BM cells into PB despite the increase of the serum level of hemopoietic/mobilizing cytokines. Thus, the physiological stress associated with major liver surgery, per se, only induced the significant mobilization of total CD34  HSCs. Moreover, we did not find any correlation between the extent of liver resection (and subsequent tissue reconstitution) and mobilization. Taken together, these findings suggest that activation of resident stem/progenitor cells may play the major role in tissue repair after liver resection, although cell therapy with HSCs may significantly help this process .

DISCLOSURES
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS
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The research was supported by the University of Bologna (funds for selected topics) and the Italian Association Against Leukemia, Bologna (BolognAil).) V) a+ G/ e8 _0 r/ d6 `
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