Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells

【摘要】  Objective- Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in ischemic tissues and that promotes neovascularization, presumably by stimulating proliferation of local endothelial cells. However, the effect of PTN on monocytes/macrophages remains unknown. We investigated the role of PTN in regulating the phenotype of monocytes/macrophages.

Methods and Results- RT-PCR, real-time PCR, and fluorescence-activated cell sorter analysis revealed that the expression of PTN by monocytic cells led to a downregulation of CD68, c-fms, and CD14 monocytic cell markers and an upregulation of FLK-1, Tie-2, vascular endothelial-cadherin, platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor, CD34, GATA-2, and GATA-3 endothelial cell markers. Fibrin gel assays showed that the treatment of mouse and human monocytic cells with PTN led to the formation of tube-like structures. In vivo studies showed that PTN-expressing monocytic cells incorporated into the blood vessels of the quail chorioallantoic membrane. The intracardial injection of PTN-expressing monocytic cells into chicken embryos showed that cells integrated only into the developing vasculature. Finally, the injection of PTN-expressing monocytes into a murine ischemic hindlimb model significantly improved perfusion of the ischemic tissue.

Conclusions- PTN expression by monocytes/macrophages led to a downregulation of their monocytic cell markers and an upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells.

Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in the highly vascularized regions of ischemic tissues. We investigated whether exposure of monocytes/macrophages to PTN alter the phenotype of these cells. Using multiple of in vitro and in vivo approaches, we found that exposure of monocytes/macrophages led to downregulation of cell phenotype and upregulation of endothelial cell characteristics. These transdifferentiated cells incorporated into newly developed vasculature and increased blood flow into ischemic hindlimb.

【关键词】  transdifferentiation pleiotrophin macrophage endothelial cell

Introduction

Neovascularization is a hallmark of chronic inflammatory diseases. By releasing a wide array of cytokines such as pleiotrophin (PTN), monocytes/macrophages play a key role in this neovascularization.

PTN is a developmentally regulated 136-aa (15.3 kDa) secreted growth/differentiation cytokine that is expressed during embryogenesis but rarely in adults (eg, few sites in the brain). PTN is differentiation or growth factor for various cell types (thus named PTN); it has mitogenic, antiapoptotic, transforming, angiogeneic, and chemotactic biological activities that can differ between its target cells. 1 Cells transformed by PTN develop into highly vascularized, aggressive tumors when implanted into nude mice (Jackson Laboratories, Bar Harbor, Me). In ischemic tissues, PTN is expressed by macrophages within an area of exuberant neovasculature that is formed at the margins of the infarct and in endothelial cells of the newly formed vessels, 2,3 suggesting a role for PTN in the neovascularization of ischemic tissue. Nothing is known about the effect of PTN on monocytes/macrophages.

Monocytes/macrophages display a high degree of plasticity, as demonstrated by their ability to transdifferentiate into endothelial cells in vitro and in vivo. 4-14 Because PTN stimulates different progenitor cells to enter lineage-specific differentiation pathway, we hypothesize that the expression of PTN by activated monocytes/macrophages in ischemic tissue may affect fate of the cells in an autocrine fashion by altering their phenotype into endothelial cells. The data presented here support this concept.

Materials and Methods

Cloning of full-length PTN, preparation of bicistronic retrovirus, primer sequences, reaction conditions, RT-PCR, real-time PCR, histology, cell culture, and fibrin gel assay are described in detail in the online supplement (available at http://atvb.ahajournals.org).

Quail Chorioallantoic Membrane Assay

The quail chorioallantoic membrane (CAM) assay used fertilized Japanese quail eggs, which were cultured ex-ovo essentially as described. 15 Fertilized Japanese quail eggs ( Coturnix coturnix japonica ) were obtained primarily from Boyd?s Bird Co. They were maintained at 37°C under ambient atmosphere, cracked in a sterile laminar flow hood at embryonic stage 3 (E3), transferred into 10-cm 2 wells of polystyrene tissue culture dishes, and cultured further at 37°C. Cells expressing PTN/green fluorescence protein (GFP) were placed on the surface of each E7 CAM; the CAMs were incubated for 3 days and then fixed. A total of 60 CAM specimens were used, 12 CAMs per group. The CAM of a fixed specimen was dissected and mounted between a glass slide and a cover slip. Fluorescent and confocal images of terminal arterial vessels from the middle region of the CAM were acquired in gray scale.

Results

Activated Monocytes/Macrophages Express PTN

PTN is expressed by activated macrophages in the ischemic rat brain. To investigate the expression of PTN by macrophages in vitro, mouse peritoneal macrophages and the mouse RAW monocytic cell line were treated with tumor necrosis factor-, and the expression of PTN was measured by Northern blot. We found that although resting monocytes/macrophages did not express PTN, their activation with tumor necrosis factor- markedly upregulated PTN expression (see online supplement), suggesting that activated macrophages express PTN, and, in turn, this cytokine can affect activity of these cells in an autocrine fashion.

PTN Downregulates the Expression of Monocytic Cell Markers

To investigate the autocrine impact of PTN expression on monocytes, human THP-1 and mouse RAW monocytic cell lines were transduced with a bicistronic retroviral vector expressing PTN and GFP (see online supplement). The presence of GFP allowed us to track the fate of the cells in vivo. The transduced cells were analyzed for the expression of monocytic cell markers by RT-PCR. Uninfected THP-1 cells ( Figure 1 A, lane 2), cells treated with phorbol 12-myristate 13-acetate ( Figure 1 A, lane 3), cells infected with GFP retrovirus ( Figure 1 A, lane 4), or PTN antisense strand ( Figure 1 A, lane 6) expressed monocytic cell markers c-fms and CD68. Retroviral transduction of cells with the PTN sense strand markedly downregulated expression of c-fms and CD68 ( Figure 1 A, lane 5). These markers were not detected in the negative control human coronary artery endothelial cells ( Figure 1 A, lane 7). GAPDH amplification showed that the RT-PCRs proceeded efficiently for all tested samples. Real-time PCR analysis confirmed the RT-PCR analysis and showed that the expression of CD68 was downregulated by 6.4 to 7.6-fold when compared with uninfected THP-1 cells, cells transduced with antisense PTN, or GFP (see online supplement). In addition, fluorescence-activated cell sorter (FACS) analysis revealed that PTN downregulated the expression of CD14 by 76%, similar to the level found in the negative control endothelial cells when compared with untransduced THP-1 cells or cells transduced with control GFP or PTN sense strand ( Figure 1 B).

Figure 1. PTN downregulates expression of monocytic cell markers. A, Total RNA was extracted from THP-1 cells grown in 10% serum (lane 2), induced to differentiate into macrophage-like cells by addition of 25 ng/mL phorbol 12-myristate 13-acetate (PMA; lane 3), transduced with retroviral bicistronic vector expressing: GFP (lane 4), PTN sense strand (lane 5), PTN antisense strand (lane 6) followed by treatment with PMA. The exponentially growing human coronary artery endothelial cells (lane 7) were used as a negative control. Analyzed monocytic cell markers were c-fms and CD-68 with primers predicted to amplify 97- and 132-bp DNA fragments, respectively. GAPDH primers were used as control a for the RT-PCR. Lane 1 is a DNA ladder marker. B, Flow cytometry analysis was performed by incubating 5 x 10 5 THP-1 cells expressing PTN or GFP with phycoerythrin-labeled anti-CD14 antibody from PharMingen. Human coronary artery endothelial cells were used as a negative control. Uninfected THP-1 cells were used as positive control, and human coronary endothelial cells (endothelial) were used as a negative control. FACS analysis was performed at the Cedars-Sinai Research Institute Core Facility. Each experiment was repeated 3 times, and each bar graph represents mean±SEM of 3 experiments.

PTN Coaxes Monocytic Cells to Acquire an Endothelial Cell Phenotype

We then asked whether PTN affects the endothelial commitment of monocytes. To explore this, the expression of endothelial cell markers in human and mouse monocytic cells that had been transduced with PTN sense, PTN antisense, or GFP were investigated by RT-PCR. The untransduced human monocytic THP-1 cells ( Figure 2 A, lane 1), mouse monocytic RAW cells ( Figure 2 A, lane 2), and human promonocytic U937 cells ( Figure 2 A, lane 3) did not express endothelial cell markers. However, cells that were transduced with the PTN sense strand ( Figure 2 A, lane 9) expressed vascular endothelial growth factor receptor-2 (FLK-1), Tie-2, vascular endothelial-cadherin (VE-cadherin), platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor (vWF), and CD34, similar to that of positive control human coronary artery endothelial cells ( Figure 2 A, lane 6). In contrast, these markers were not detected in THP-1 cells transduced with the PTN antisense strand ( Figure 2 A, lane 10) or the GFP control vector ( Figure 2 A, lane 11). Likewise, endothelial cell markers were not detected in nonmonocytic cells, such as NIH 3T3 cells ( Figure 2 A, lane 4), human coronary artery smooth muscle cells ( Figure 2 A, lane 5), RPMI 8226 B lymphocyte plasmacytoma cell line ( Figure 2 A, lane 7), and human skin fibroblasts ( Figure 2 A, lane 8). The weak expression of FLK-1 in smooth muscle cells ( Figure 2 A, lane 5) is consistent with the expression of this endothelial cell marker in human smooth muscle cells. 16

Figure 2. PTN upregulates expression of endothelial cell markers in the monocytic cells. A, RT-PCR analysis of endothelial cell markers. Total RNA isolated from untransduced and transduced cells were used for RT-PCR analysis, using specific primers for endothelial cell markers (see online supplement). To ensure semiquantitative results of the RT-PCR analysis, the number of PCR cycles for each set of primers was checked to be in the linear range of the amplification. In addition, all RNA samples were adjusted to yield equal amplification of GAPDH as an internal standard. The amplified products were separated on 1.2% agarose gels and stained with ethidium bromide. B, A representative flow cytometry analysis of v ß 3 integrin expression by the monocytic cells. GFP (top left panel) or PTN-expressing THP-1 cells (bottom left panel) were incubated with a 1:100 dilution of anti-human v ß 3 mouse antibody (Chemicon Co). After washing, cells were incubated with 1:500 dilution of phycoerythrin-labeled anti-mouse antibody (Sigma), fixed, and analyzed by FACS, as described above. In addition, human coronary artery endothelial cells in the absence (top right panel) or presence of anti- v ß 3 antibody (bottom right panel) were used as a positive control. C, RT-PCR analysis of transcription factors. The PCR was performed as described in A, and the primers and PCR condition are described in the online supplement.

Real-time PCR analysis was used to compare the expression level of the VE-cadherin, vWF, and platelet endothelial cell adhesion molecule-1 genes in the PTN-transduced THP-1 cells with those of positive control human endothelial cells. The expression levels of these genes were 0.8 x 10 5, 2.9 x 10 5, and 1.3 x 10 5 copies/100 ng of RNA, respectively ( P value all <0.001). These levels of expression are similar to those of the positive control human endothelial cells (0.6 x 10 5, 3.2 x 10 5, and 1.4 x 10 5 copies/100 ng endothelial cell RNA; P value all <0.001, respectively).

This phenotypic modulation of monocytic cells by PTN was further substantiated by investigating the expression of endothelial cell-specific v ß 3 integrin by flow cytometry. FACS analysis revealed that 80±4% of THP-1 cells expressing PTN are positive for v ß 3 ( Figure 2 B, bottom left panel) compared with 1±3% of THP-1 cells expressing GFP ( Figure 2 B, top left panel). The level of v ß 3 integrin in the PTN-expressing THP-1 cells (80±4%) is similar to those of human coronary artery endothelial cells ( Figure 2 B, bottom right panel). The omission of the anti- v ß 3 antibody reduced positivity to 4±3% ( Figure 2 B, top right panel). Further, FACS analysis revealed that 72±5% of THP-1 cells transduced with the PTN sense strand expressed Tie-2 compared with 7±3% of cells transduced with GFP retroviral vector.

PTN Mediates the Phenotypic Modulation of Monocytes at the Transcriptional Level

Next, we asked whether the phenotypic modulation of monocytic cells by PTN is regulated at the transcriptional level. To accomplish this, the expression of the transcription factors GATA-2 and GATA-3 that affects endothelial cell commitment 17-20 was measured. RT-PCR analysis showed that untransduced THP-1 ( Figure 2 C, lane 1), monocytic RAW ( Figure 2 C, lane 2), and U937 cells ( Figure 2 C, lane 3) as well as THP-1 cells transduced with either the PTN antisense strand ( Figure 2 C, lane 10) or the GFP control vector ( Figure 2 C, lane 11) did not express these transcription factors. In contrast, THP-1 cells infected with the PTN sense strand ( Figure 2 C, lane 9) expressed both GATA-2 and GATA-3 similar to the control human coronary artery endothelial cells ( Figure 2 C, lane 6). Nonmonocytic cells such as mouse NIH 3T3 cells ( Figure 2 C, lane 4), smooth muscle cells ( Figure 2 C, lane 5), RPMI 8226 B lymphocyte plasmacytoma cells ( Figure 2 C, lane 7), and human dermal fibroblasts ( Figure 2 C, lane 8) did not express GATA-2 and GATA-3. Figure 2 of the supplement further demonstrates the colocalization of GATA-2 expression and VE-cadherin in the transdifferentiated cells.

The monocytic cell lines that we used (THP-1 and RAW cells) are established cell lines with known monocytic cell characteristics that do not exhibit characteristics of multipotent cells. The expression of differentiated cell markers such as CD68, c-fms, and CD14 in THP-1 cells support this concept. However, to investigate the maturity/immaturity of THP-1 cells and RAW cells in more detail the expression of AC133 and Oct-4, 2 well-known markers of stem/progenitor cells 21-23 were studied ( Figure 2 C). Among the examined cells, AC133 was expressed only in human U937 cells ( Figure 2 C, lane 3), and Oct-4 was expressed in mouse NIH 3T3 cells ( Figure 2 C, lane 4) and human RPMI 8226 B lymphocyte plasmacytoma cells ( Figure 2 C, lane 7).

PTN Induces Functional Transdifferentiation of Monocytic Cells Into Endothelial Cells

In Vitro Studies

To determine whether the transdifferentiated cells function like endothelial cells, they were cultured in a fibrin gel assay and tube formation was investigated. This showed that cultured THP-1 cells expressing PTN ( Figure 3 A) or RAW cells expressing PTN ( Figure 3 C) formed capillary-like structures. In contrast, the morphology of THP-1 cells ( Figure 3 B) or RAW cells ( Figure 3 D) that expressed GFP did not change.

Figure 3. PTN coax monocytic cells to form tubular structures in vitro. Fibrin gels were prepared essentially as described. 26 THP-1 cells (5 x 10 5 ) expressing PTN (A) or GFP (B) or RAW cells expressing PTN (C) or GFP (D) were seeded on the fibrin matrix, incubated at 37°C, and the formation of tubular structures was analyzed by phase-contrast microscopy. Primary mouse peritoneal cells were isolated by standard methods using 10- to 12-week-old BALB/C mice. The nonadherent cells were removed after 3 and 7 days of culture, and the cells were further cultured for 14 days, at which time conditioned media (100 µL) derived from RAW cells expressing PTN/GFP (E) or GFP (F) were added to cells for 48 hours. For the adsorption experiment, the conditioned media were preincubated with 20 µg of a goat anti-human PTN polyclonal antibody (Calbiochem; H) or goat IgG (G) at 37°C for 30 minutes. The media were then incubated with agarose-anti-goat antibody for 30 minutes at room temperature to remove the complex before addition to the cells.

To determine whether PTN affects the phenotype of primary monocytes, peritoneal macrophages were isolated from mice and cultured in a fibrin gel in the presence of conditioned media derived from either RAW cells expressing PTN/GFP or GFP. PTN induced the formation of capillary-like structures in the primary cells ( Figure 3 E), whereas cells exposed to the GFP-derived media did not form such structures ( Figure 3 F). To assess whether the activity found in the conditioned media is specifically related to PTN, the media were preabsorbed with an anti-PTN neutralizing antibody or an isotype-matched nonspecific antibody before its addition to cells. The nonspecific antibody had no effect on capillary formation ( Figure 3 G), whereas the anti-PTN neutralizing antibody markedly downregulated capillary formation ( Figure 3 H).

In Vivo Studies

The quail CAM was used to determine whether the transdifferentiated cells incorporate into blood vessels. Fertilized Japanese quail eggs were cultured ex-ovo at E3, and then RAW cells expressing PTN were transplanted onto the surface of the CAM at E7 (see online supplement). RAW cells or 293 cells expressing GFP were used as controls in addition to PBS. After 3 days, at E10 (see online supplement), CAMs were analyzed by fluorescence and confocal microscopy.

RAW cells expressing PTN/GFP integrated into large and small CAM blood vessels at various branches in 9 of 12 quail embryos ( Figure 4 A; see online supplement). In contrast, RAW cells expressing GFP did not get incorporated into any blood vessels of the CAM ( Figure 4 B). To further demonstrate the specificity of the incorporation of RAW cells, the confocal image of the newly formed fluorescent-labeled blood vessels was overlapped with a differential interference contrast image. This improved the contrast, and the incorporation of cells into the blood vessels was examined in the context of a full view of the CAM vasculature. Although the expression of GFP per se was insufficient for the incorporation of RAW cells into the CAM ( Figure 4 C), expression of PTN was sufficient for the integration of cells into CAM vasculature ( Figure 4 D). This integration generated chimeric quail-mouse blood vessels.

Figure 4. PTN-expressing monocytic cells specifically incorporate into blood vessels. RAW cells (1 x 10 6 ) expressing PTN/GFP (A and D) or GFP (B and C) were implanted onto the surface of E7 quail CAM and then were incubated further at 37°C for 72 hours, at which time they were fixed in 4% paraformaldehyde/2% glutaraldehyde/PBS. Fluorescent and confocal images of terminal arterial vessels from the middle region of the CAM were acquired in gray scale at a resolution of 13 µm per pixel. The fixed CAMs with fluorescence images overlapped with differential interference contrast (C and D) images at x 4 magnification.

However, the CAM assay did not reveal whether the cells incorporated into already established or developing blood vessels. To determine this, RAW cells expressing either PTN/GFP or GFP were injected intracardially into stage 16-17 chicken embryos. At this stage, the chicken brain and ocular system are being developed, whereas their cardiovascular system is already established. The embryos were collected 2 to 3 days after injection, fixed, and sectioned (n=10 embryos/cell types). The sections were stained with either anti-GFP or anti-Tie-2 antibodies. In embryos injected with RAW cells expressing PTN/GFP, most of the positive staining appeared along the developing vessels in the head, eyes, and intersomitic regions ( Figure 5, PTN panel). In contrast, embryos injected with GFP-expressing RAW cells did not stain ( Figure 5, GFP panel). This demonstrates that only the RAW cells expressing PTN had the ability to incorporate into developing blood vessels.

Figure 5. PTN-expressing cells specifically integrate into developing vasculature. PTN/GFP- or GFP-expressing RAW cells (1 to 2 x 10 5 cells in 2 to 4 µL PBS) were injected into the hearts of stage 16-17 chick embryos (10 embryos/cell types). Embryos were killed 2 to 3 days after injection, fixed, embedded in OTC, and frozen sections were cut and stained with anti-GFP polyclonal antibodies (Santa Cruz Biotechnology) or anti-Tie-2 antibodies. The immunopositivity (brown color) was observed in embryos injected with RAW cells expressing PTN/GFP. The positivity localized along the developing blood vessels in the head, eyes, and intersomitic region (right panel). In contrast, immunostaining of embryo injected with RAW cells expressing the GFP gene show no staining (left panel). In some cases, faint staining was detected around the amniotic cavity.

Finally, the ability of transdifferentiated cells to improve blood flow into ischemic tissue was measured. Hindlimb ischemia was induced in BALB/C mice followed by the injection of RAW cells expressing PTN 1 day after surgery. Laser Doppler perfusion imaging monitored blood flow at days 7, 14, and 21 after surgery. BALB/C background mice were selected for these experiments because RAW cells are congenic to this mouse strain, thus avoiding a potential graft-versus-host complication. Figure 6 A shows that blood flow recovery at 7 days after surgery was significantly higher in mice injected with PTN-expressing RAW cells when compared with control mice injected with GFP-expressing RAW cells. Fluorescence images of frozen sections from the mice injected with the PTN/GFP-RAW cells show that the cells were incorporated into blood vessels of ischemic hindlimb ( Figure 6 B), whereas such incorporation was not detected in the control mice (data not shown). Cumulative laser Doppler perfusion imaging data show that mice injected with cells expressing PTN had 60±3% increase in blood flow at 7 days, 50±4% at 14 days, and 30±3% at 21 days compared with control animals injected with PBS at similar time points ( Figure 6 C).

Figure 6. PTN-expressing monocytic cells improve blood flow into ischemic murine hindlimb. BALB/C male mice (Jackson Laboratories; 20 to 25 g) were sedated, and the left femoral artery was ligated with 8-0 silk sutures at its proximal origin from the iliac artery and distally at the bifurcation into the popliteal and saphenous arteries to induce mild ischemia. RAW cells expressing PTN/GFP (1 x 10 6 cells in 200 µL PBS per mouse) were injected intravenously (tail vein) 24 hours after surgery (8 mice per group). Mice injected with 200 µL of PBS were used as controls (8 mice per group). Repeated hindlimb blood flow measurements over the region of interest (from the patella to the midfoot) were obtained at baseline, immediately after surgery, and serially over 3 weeks by laser Doppler perfusion imaging. Perfusion is expressed as a ratio of right (ischemic) to left (normal) limb. Representative color-coded images (red is highest velocity, green intermediate, and blue, lowest velocity), which reflect red blood cell velocity at day 7 after surgery, are shown (A). The top 4 panels show BALB/C mice injected with PBS, and the bottom 4 panels show animals injected with RAW cells expressing PTN/GFP. B, The immunofluorescence image of the frozen section from the ischemic hindlimb 7 days after injection of PTN/GFP cells shows that the cells are incorporated into the artery and veins. C, Cumulative results for the groups of mice monitored for 21 days after surgery are shown graphically as a ratio of blood flow in ischemic limb to that in the nonischemic limb at each time point. The Student t test analysis showed that the increase in the blood flow is statistically significant in the mice group injected with PTN/GFP compared with control groups at all the timer points examined. Values are mean±SE ( P <0.05 are considered statistically significant).

Discussion

PTN is produced by activated monocytes in the highly vascularized region of ischemic brain and is thought to promote angiogenesis by stimulating sprouting/proliferation of endothelial cells. However, nothing is known about the autocrine effect of PTN in ischemic tissues (ie, its effect on monocytes/macrophages). These cells exhibit a high degree of plasticity, as demonstrated by their ability to alter their phenotype into functional endothelial cells. We found that activated monocytes/macrophages express PTN, and, in turn, this cytokine interacts with cells leading to the downregulation of monocytic cells markers and upregulation of fully differentiated endothelial cell markers. Real-time PCR analysis showed that the level of expression of these markers is similar to those found in the human coronary artery endothelial cells, suggesting that the PTN-mediated upregulation of endothelial markers is functionally relevant. This idea is further underscored by the expression of v ß 3, an integrin that is required for the interaction of endothelial cells with the matrix and the formation of tube-like structures. The fibrin gel assay confirmed this and further supported the notion that the transdifferentiated cells exhibit the characteristics of functional endothelial cells in vitro. In vivo studies bore out our in vitro findings and show that the transdifferentiated mouse cells incorporate into blood vessels. In the CAM assay, the RAW cells were implanted onto the CAM, which allowed them to distribute throughout the CAM, meaning that the cells had the opportunity to randomly incorporate into any part of the CAM structure. However, they specifically integrated into the CAM vasculature, suggesting that the cells had all the necessary information required for homing into the vascular tree. The differential interference contrast image further supports this notion and further showed that the homing and integration of the implanted cells was specific to the CAM blood vessels. Chicken embryo experiments further supported this specificity of integration and further demonstrated that although the intracardially injected cells distributed throughout the developing embryo, PTN-expressing cells incorporated only into the developing blood vessels of the brain and eye but not into the already established cardiovascular system of the embryo. This ability of transdifferentiated mouse cells to incorporate into developing blood vessels also appears to be a conserved phenomenon because similar results have been reported with human peripheral blood monocytes in which ex vivo-expanded purified CD14 cells exhibited endothelial cell characteristics in vitro and were incorporated into newly formed blood vessels in vivo. 6

This PTN-induced transdifferentiation appears to be regulated at the transcriptional level because both the GATA-2 and GATA-3 transcription factors that are known to regulate the endothelial cell markers are also upregulated by PTN in monocytic cells, suggesting that this phenotypic alteration may be related to the nuclear reprogramming of the monocytic cells. This transcriptional regulation of the endothelial commitment of monocytic cells by PTN may not be related to the pluripotency characteristics of cells because both THP-1 and RAW cells did not express CD133 and Oct-4, 2 well-known markers of stem/progenitor cells. In addition, primary mouse peritoneal macrophages acquired endothelial cell characteristics when exposed to PTN. Together, these data suggest that PTN has the ability to alter the phenotype of fully differentiated monocytes/macrophages into fully differentiated endothelial cells. This phenotypic modulation is consistent with the classical definition of transdifferentiation (ie, an alteration of the phenotype of 1 fully differentiated cell type into another fully differentiated phenotype). 24

The ability of monocytes/macrophages to transdifferentiate into endothelial cells does not seem to be related to their proliferative state. Resting primary human peripheral blood monocytes are known to transdifferentiate into endothelial cells. 4-14,25 Similarly, we found that PTN induces transdifferentiation of mouse peritoneal cells, a nonproliferating cell type. The proliferating human THP-1 cells and mouse RAW cells also transdifferentiated into endothelial cells. Together, these data suggest that the ability of monocytes to transdifferentiate into endothelial cells is independent of their proliferative activity, suggesting that tissue macrophages found in chronically inflamed tissues may be able to transdifferentiate into endothelial cells in the presence of PTN.

In addition to primary cells, we used THP-1 and RAW clonal cells to investigate the transdifferentiation activity of PTN for 2 reasons. First, these cells are replicating, and therefore they allowed us to label them with GFP and track their fate in vivo. Second, these cells are a homogeneous population of differentiated cells, and they are not contaminated with other cell types. This allowed us to exclude the potential contribution of contaminating cells to the PTN-induced transdifferentiation of monocytes, a possibility that cannot be excluded when primary monocytes are used. In addition, the use of clonal cells excludes cell fusion between 2 cell types as a potential mechanism for PTN-mediated transdifferentiation.

Monocytes/macrophages are known to transdifferentiate into endothelial cells. However, factor(s) that regulate this event remain unknown. PTN is expressed by activated monocytes/macrophages in the highly vascularized regions of ischemic brain. We offered evidence that PTN produced by macrophages interacts with them in an autocrine fashion, coaxing the cells to transdifferentiate into functional endothelial cells. These transdifferentiated cells have the ability to increase blood flow into ischemic tissue. Thus, in addition to previously reported angiogenic activity, our data identified a novel activity for PTN that could be partly responsible for the neovascularization of inflamed tissues.

Acknowledgments

This work was supported by grants from the National Institutes of Health (ROI HL50566), Eisner Foundation, Heart Funds, and Women?s Cancer Funds of Entertainment Industry Fund. B.G.S. is an established investigator of American Heart Association.

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作者单位：Atherosclerosis Research Center (B.G.S., Z.Z., L.W., L.S., H.C., M.Q., P.K.S.), Division of Cardiology, and Department of Surgery (M.R.S.-H., S.W.-H.), Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, Calif.